Acetylenedicarboxyl linkers and their uses in specific conjugation of a cell-binding molecule

ABSTRACT

Cell binding agent-drug conjugates comprising bridge linkers, and methods of using such linkers and conjugates are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/799,666,filed on Jul. 15, 2015, entitled “ACETYLENEDICARBOXYL LINKERS AND THEIRUSES IN SPECIFIC CONJUGATION OF A CELL-BINDING MOLECULE.” The content ofthe prior application is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the preparation of novel linkers usedfor the specific conjugation of compounds, in particular, cytotoxicagents to a biological molecule. The present invention also relates tomethods of making cell-binding agent-drug (cytotoxic agent) conjugatesin a specific manner comprising either modification of drugs with theselinkers first, followed by reaction with prepared cell-binding agents;or modification of cell-binding agents with these linkers first,followed by reaction with drugs.

BACKGROUND OF THE INVENTION

Proteins, specifically antibodies have been extensively used intherapeutic applications, in vitro assays as research reagents and invivo as diagnostic tools or as therapeutic drugs (Gad, S. C. Drugdiscovery handbook, published by Wiley-Interscience, 2005). For manyapplications the protein needs to be modified with an interesting group,such as a cytotoxic drug, a radio label element or a chromphore moleculefor use in therapy or a detection agent when used in diagnostics(Teicher, B. A. et al. Clin. Cancer Res. 2011, 17, 6389-97; Elsadek, B.et al., J. Control Release, 2012, 157, 4-28). One of these applications,called antibody-drug conjugates (ADCs), which is the exquisite targetingability of antibodies in combination with the cytotoxic action ofanticancer agents, enables to target and deliver drugs to cancer cellsleaving normal cells largely unaffected, has been intensely exploitationin the last two decades. In particular, since US FDA approvals ofAdcetris (brentuximab vedotin) in 2011 and Kadcyla (ado-trastuzumabemtansine) in 2013, the applications of antibody-drug conjugate (ADC) asa promise targeted treatment of cancers have been exploded and almostevery major pharmaceutical and biotech company has adopted this approach(Chari, R. et al, Angew. Chem., Int. Ed. 2014, 53, 3796-3827; Sievers,E. L. et al. Annu Rev Med. 2013, 64, 15-29; Mehrling, T. Future Oncol,2015, 11, 549). Currently there are more than 50 ADC drugs in the clinictrials according to www.clinictrails.gov.

The first-generation ADCs, including Kadcyla and Adcetris, are producedthrough nonselective conjugation of native lysine amines or interchaincysteine thiols on an antibody respectively to a cytotoxic drug. Sincethere are over 50 surface-exposed lysines and 8 hinge cysteine residuesin IgG1 antibodies, this nonselective conjugation results in randomlycross-linkage of cytotoxic drugs to practically all areas of theantibody molecule, particularly having a diverse population of ADCs witha wide distribution of drugs per antibody (DAR) (Wang, L., et al. 2005Protein Sci. 14, 2436; Hamblett, K. J., et al. 2004 Clin. Cancer Res.10, 7063). Thus some of the undesired ADC subpopulation could lead toshorter circulation half-life, lower efficacy, potentially increasedoff-target toxicity and a wide range of in vivo pharmacokinetic (PK)properties (Hamblett, K. J. et al, Clin. Cancer Res. 2004, 10,7063-7070; Adem, Y. T. et al, Bioconjugate Chem. 2014, 25, 656-664;Boylan, N. J. Bioconjugate Chem., 2013, 24, 1008-1016; Strop, P., et al2013 Chem. Biol. 20, 161-167). In addition, with this classicalconjugation, the batch-to-batch consistency in ADC production can bechallenging and may require diligent manufacturing capabilities(Wakankar, A. mAbs, 2011, 3, 161-172).

Therefore, biotechnology companies and academic institutions are highlyfocusing on establishing novel reliable methods for site-specific ADCconjugation. So far, there are several approaches developed in recentyears for site selective ADC preparation (Panowski, S, 2014, mAbs 6,34). They include incorporation of unpaired cysteines, e.g. engineeredreactive cysteine residues, called THIOMAB from Genentech (Junutula, J.R., et al 2010 Clin. Cancer Res. 16, 4769; Junutula, J. R., et al 2008Nat Biotechnol. 26, 925-32; U.S. Pat. Nos. 8,309,300; 7,855,275;7,521,541; 7,723,485, WO2008/141044), genetically introduced glutaminetag with Streptoverticillium mobaraense transglutaminase (mTG) (Strop,P., Bioconjugate Chem., 2014, 25, 855-862; Strop, P., et al., 2013,Chem. Biol. 20, 161-167; U.S. Pat. No. 8,871,908 for Rinat-Pfizer) orwith Microbial transglutaminase (MTGase) (Dennler, P., et al, 2014,Bioconjug. Chem. 25, 569-578. US pat appl 20130189287 for Innate Pharma;U.S. Pat. No. 7,893,019 for Bio-Ker S.r.l. (IT)), incorporation ofthiolfucose (Dennler, P., et al, 2014 Bioconjugate Chemistry 25, 569;Okeley, N. M., et al 2013 Bioconjugate Chem. 24, 1650), incorporation ofunnatural amino acids through mutagenesis (Axup, J. Y., et al., 2012,Proc. Natl. Acad. Sci. 109, 16101-16106; Zimmerman, E. S., et al., 2014,Bioconjug. Chem. 25, 351-361; Wu, P., et al, 2009 Proc. Natl. Acad. Sci.106, 3000-3005; Rabuka, D., et al, 2012 Nat. Protoc. 7, 1052-67; U.S.Pat. No. 8,778,631 and US Pat Appl. 20100184135, WO2010/081110 for SutroBiopharma; WO2006/069246, 2007/059312, U.S. Pat. Nos. 7,332,571,7,696,312, and 7,638,299 for Ambrx; WO2007/130453, U.S. Pat. Nos.7,632,492 and 7,829,659 for Allozyne), Incorporation of selenocysteineinto antibodies (Hofer, T., et al 2009, Biochemistry 48, 12047-12057;U.S. Pat. No. 8,916,159 for US National Cancer Institute), Convertion ofcysteines located in the CXPXR consensus sequence to formylglycine(FGly) with formylglycine generating enzyme (FGE) (Drake, P. M., et al.,2014, Bioconjug. Chem. 25, 1331-1341. Carrico; Isaac S. et al U.S. Pat.Nos. 7,985,783; 8,097,701; 8,349,910, and US Pat Appl 20140141025,20100210543 for Redwood Bioscience), and through glycoengineeringlyintroduction of sialic acid with the use of galactosyl- andsialytransferases (Zhou, Q., et al 2014, Bioconjug. Chem., 25, 510-520,US Pat Appl 20140294867 for Sanofi-Genzyme). These above methods haveproduced nearly homogeneous product profiles, but they are requiredantibody-engineering processes and reoptimization of cell cultureconditions. Moreover, expression yields for genetic encoding of anunnatural amino acid were typically not promisingly high enough (Tian,F., et al, 2014, Proc. Natl. Acad. Sci. U.S.A 111, 1766-71) which has asignificant impact on the cost of goods of the ADC. In addition, it hasbeen known that ADCs obtained by conjugation to cysteine side chainsoften display limited stability in circulation, leading to prematuredisconnection of the cytotoxic payload before the tumor site is reached(Junutula, J. R., et al 2008, Nat. Biotechnol. 26, 925-32).

The disulfide bond structures of the four subclasses of IgG antibodieswere known in the 1960s (Milstein C. Biochem J 1966, 101:338-351; Pink JR, Milstein C. Nature 1967, 214:92-94; Frangione B, Milstein C. Nature1967, 216:939-941; Pink J R, Milstein C. Nature 1967, 216:941-942;Frangione B, et al. Biochem J 1968, 106, 15-21; Frangione B, Milstein C.J Mol Biol 1968; 33:893-906; Edelman G M, et al. Proc Natl Acad Sci USA1969; 63:78-85; Frangione B, et al. Nature 196, 221:145-148,Spiegelberg, H. L. et al Biochemistry, 1975, 10, 2157-63). Disulfidebond structure is critical for the structure, stability, and biologicalfunctions of IgG molecules. Among the four subclasses of IgG antibodies,IgG₁, IgG₂, IgG₃ and IgG₄, each IgG contains a total of 12 intra-chaindisulfide bonds; each disulfide bond is associated with an individualIgG domain. The two heavy chains are connected in the hinge region by avariable number of disulfide bonds: 2 for IgG₁ and IgG₄, 4 for IgG₂ and11 for IgG₃. The light chain of the IgG₁ is connected to the heavy chainby a disulfide bond between the last cysteine residue of the light chainand the fifth cysteine residue of the heavy chain. But, for IgG₂, IgG₃and IgG₄, the light chain is linked to the heavy chain by a disulfidebond between the last cysteine residue of the light chain and the thirdcysteine residue of the heavy chain (Liu, H. and May, K., 2012, mAbs 4,17-23). On the ranks of the susceptibility of disulfide bonds in humanIgG1 antibodies by experimental reduction, differential alkylation, andLC-MS analysis (Liu, H, et al Anal. Chem., 2010, 82, 5219-5226), interchain disulfide bonds are more susceptible to reduction than intra chaindisulfide bonds, and the disulfide bonds between the light chain andheavy chain were more susceptible than disulfide bonds between the twoheavy chains. The upper disulfide bond of the two inter heavy chaindisulfide bonds was more susceptible than the lower one. Furthermore,disulfide bonds in the CH2 domain were the most susceptible toreduction. Disulfide bonds in VL, CL, VH, and CH1 domains had similarand moderate susceptibility, while disulfide bonds in the CH3 domainwere the least susceptible to reduction (Liu, H, et al Anal. Chem.,2010, 82, 5219-5226).

Based on the more susceptibility of inter chain disulfide bonds in humanIgG1 antibodies, several institutions and companies adopted thechemically specific conjugation strategy through rebridging reducedinterchain disulfide bonds of a native antibody, such as, using bromo ordibromo-maleimides, called next generation maleimides (NGMs)(Schumacher, F. F., et al 2014, Org. Biomol. Chem. 12, 7261-7269; UCLCancer Institute), applying bis-alkylating reagents via a three-carbonbridge (Badescu, G., et al., 2014, Bioconjug. Chem. 25, 1124-1136.,WO2013/190272, WO2014/064424 for PolyTherics Ltd), with di-substitutedheteroaryl bridge (US Pat Appl. 2015/0105539 for Concortis Biosystem),or through di-maleimide as a bridge (WO2014/114207). We have also usedbromo maleimide and dibromomaleimide linkers to conjugate both drugs andantibodies for a quite while (WO2014/009774, PCT/IB2012/053554).However, these above bridge linkers were designed in the way toconjugate only one cytotoxic agents to a pair of disulfide bonds, andtherefore at most of time they only produced ADCs at DAR less than 2(drugs per antibody), due to limited numbers (about two pairs) ofreduced disulfide bonds are more accessible for conjugation.

As one of the major issues for ADCs is the limited numbers or amount ofcytotoxic compound that ultimately reaches the tumor, and the favorableDAR over 3 is much important factor for improvement of ADC therapeuticalindex (Epenetos, A. A. et al, Cancer Res., 1986, 46, 3183-3191; Chari,R. V. Acc. Chem. Res., 2008, 41, 98-107, Zhao, R. Y. et al, 2011, J.Med. Chem. 54, 3606-3623), we therefore disclose novelacetylenedicarboxyl linkers of this invention that not only are able toconjugate two or more drugs per linker for achieving higher DARs (≧4),but also can selectively rebridge pairs of reduced inter chain disulfidebonds on surface of antibody due to the nature of stretch-out triplebond of the acetylenedicarboxyl group, particularly when two cytotoxicagents are linked at both ends of the stretch-out bridge linker, forminga quite large size (>20 Å) of molecule which in turn hardly accesses tothe other disulfide bond sites, such as reduced intra chain disulfidebonds beneath the antibodies. The acetylenedicarboxyl linkers of thisinvention therefore can be used for selective bridging the pairs of freethiols on the inter chain of antibody, which are generated by overloadedTCEP or DTT, and for producing an ADC having DAR (drugs per antibody)over four. And the over reduced pairs of thiol groups that areinaccessibly reached by the bridge linkers, particularly by thestretch-out acetylenedicarboxyl linkers containing two cytotoxic agents,can be recoupled (regenerated) by an oxide, e.g. dehydroascorbic acid(DHAA) or Cu(II), to form back disulfide bonds at the end ofconjugation. In a word, these bridge linkers of the invention can makehomogeneous production of specific ADCs in a simple manner.

SUMMARY OF THE INVENTION

The present invention provides linkers containing anacetylenedicarboxylic group to link two drugs to a cell-binding agent(e.g., an antibody). The preferred formula of the cell-bindingmolecule-linker-drug conjugates can be represented as:

wherein Cb is a cell-binding agent, L is a acetylenedicarboxyl linker,Drug1 and Drug2 are a drug molecule, n is an integer from 1 to 20, andtwo S (sulfur) elements from Cb bridgely link to L, which covalentlyconnects two or more drugs (per bridge linker L). The advantages inapplying the linker in the cell molecule-drug conjugate are: a).Retaining the stability of the conjugates by covalently cross-linking(rebridging) the pairs of reduced disulfur atoms of the cell-bindingagents, particularly of antibodies; b). Enabling conjugation of thecytotoxic agents/drugs to specific sites of a cell-binding molecule,e.g. the inter chain disulfide bond sites of IgG antibodies, resultingin homogeneous production of ADC.

In one aspect of the present invention, the linker is represented byFormula (I)

Wherein the acetylenedicarboxyl group on the linker is capable ofreacting with a pair of sulfur atoms of the cell-binding agent. Thesulfur atoms are preferred pairs of thiols reduced from the interchaindisulfide bonds of the cell-binding agent by a reducing agent, such asDTT and/or TCEP;

Z₁ and Z₂ are the same or different a function group that enables toreact with a cytotoxic drug, to form a disulfide, ether, ester,thioether, thioester, peptide, hydrazone, carbamate, carbonate, amine(secondary, tertiary, or quartary), imine, cycloheteroalkyane,heteroaromatic, alkoxime or amide bond;

R₁ and R₂ are the same or different, and are absent, linear alkyl havingfrom 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1-6carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula(OCH₂CH₂)_(p), wherein p is an integer from 0 to about 1000, orcombination thereof.

Additionally R₁ and R₂ are respectively a chain of atoms selected fromC, N, O, S, Si, and P, preferably having 0-500 atoms, which covalentlyconnects to X₁ or X₂ and Z₁ or Z₂. The atoms used in forming the R₁ andR₂ may be combined in all chemically relevant ways, such as formingalkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters,amines, imines, polyamines, hydrazines, hydrazones, amides, ureas,semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes,amino acids, peptides, acyloxylamines, hydroxamic acids, or combinationthereof.

X₁ and X₂ are independently selected from NH, N(R₃), O, S or CH₂; R₃ isH, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkylhaving from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl oralkynyl, or 1˜6 carbon atoms of esters, ether, amide, or polyethyleneoxyunit of formula (OCH₂CH₂)_(p), wherein p is an integer from 0 to about1000, or combination thereof.

In another aspect, this invention provides a cell-binding agent-drugconjugate of Formula (II), in which the cell-binding agent, Cb, and thedrug, Drug1 and Drug2, have reacted at the ends of the bridge linker:

wherein:

Cb represents a cell-binding agent, preferred an antibody;

Inside the bracket (parentheses) are the linker-drug components that areconjugated to pairs of sulfur atoms of the cell-binding molecule. Thesulfur atoms are preferred pairs of thiols reduced from the interchaindisulfide bonds of the cell-binding agent by a reduction agent, such asDTT and/or TCEP;

Drug₁ and Drug₂ represent the same or different cytotoxic agents, whichlinked to the cell-binding agent via the bridge linker by a disulfide,thioether, thioester, peptide, hydrazone, ether, ester, carbamate,carbonate, cycloheteroalkyane, heteroaromatic, alkoxime or amide bond;

n is 1˜20; R₁, R₂, X₁ and X₂ are described the same previously inFormula (I).

In a further aspect, the present invention provides a modifiedcell-binding agent of Formula (III), in which the cell-binding agent,Cb, through its pair of thiols generated with reduction of disulfidebonds, has reacted with the bridge linker, which has Z₁ and Z₂, thefunction groups capable of reacting with a drug:

Wherein Cb, Z₁, Z₂, n, R₁, R₂, X₁, and X₂ are defined the same as inFormula (I) and (II).

In an even further aspect, the present invention provides a modifieddrug of Formula (IV), in which the drug, Drug₁ and Drug₂, have reactedwith the linker of Formula (I), which still has the acetylenedicarboxylgroup capable of reacting with a pair of sulfur atoms of thecell-binding agent:

Wherein Drug₁, Drug₂, R₁, R₂, X₁, and X₂ are defined the same as inFormula (I) and (II).

The present invention further relates to a method of making acell-binding molecule-drug conjugate of Formula (II), wherein the drugs,Drug₁ and Drug₂ are linked to a cell-binding agent via the bridgelinker.

The present invention also relates to a method of making a modifiedcell-binding molecule of Formula (III), wherein the cell-bindingmolecule is reacted with the bridge linker of Formula (I).

The present invention also relates to a method of making a modified drugof formula (IV), wherein the drug is reacted with the bridge linker ofFormula (I).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of a bridge linker containing polyethyleneglycol groups and the application of this linker in the conjugation ofan antibody with drugs.

FIG. 2 shows the synthesis of a bridge linker and the application ofthis linker in the conjugation of drugs to an antibody via oximelinkage.

FIG. 3 shows the synthesis of a bridge linker containing a peptide andthe application of this linker in the conjugation of drugs to anantibody via amide linkage.

FIG. 4 shows the synthesis of a bridge linker containing peptides,polyethylene glycol.

FIG. 5 shows the synthesis of a bridge linker containing peptides andpolyethylene glycols, and the application in the conjugation of two orfour drugs per linker to an antibody via amide linkage.

FIG. 6 shows the synthesis of tubulysin analogs which are modified withthe bridge-linker containing peptides and polyethylene glycols.

FIG. 7 shows the synthesis of the conjugates of cell-bindingmolecule-tubulysin analogs via the bridge-linker containing polyethyleneglycols.

FIG. 8 shows the synthesis of the conjugates of cell-bindingmolecule-maytansinoids via the bridge-linker.

FIG. 9 shows the synthesis of the conjugates of cell-bindingmolecule-MMAF analogs via the bridge-linker.

FIG. 10 shows the synthesis of the conjugates of cell-bindingmolecule-tubulysin analogs via the bridge-linker.

FIG. 11 shows the comparison of the anti-tumor effect of conjugatecompounds 91 and 93 with T-DM1 using human gastric tumor N87 cell modelat dosing, 5 mg/kg, i.v., one injection. Both compounds 91 and 93 werebetter than T-DM1: the compound 93 completely eradicated the tumor atday 14˜18 till day 60 (the end of experiment). Compound 91 eradicatedthe tumor at day 14˜22 until day 42˜50 and inhibited the tumor growthtill the end of the test. In contrast T-DM1 did not completely eliminatethe tumor and only inhibited the tumor growth for 44 days.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” refers to an aliphatic hydrocarbon group which may be straightor branched having 1 to 8 carbon atoms in the chain. “Branched” meansthat one or more lower C numbers of alkyl groups such as methyl, ethylor propyl are attached to a linear alkyl chain. Exemplary alkyl groupsinclude methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl,3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl,3-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from —C₁-C₈ alkyl and aryl. “Halogen”refers to fluorine, chlorine, bromine or iodine atom; preferablyfluorine and chlorine atom.

“Heteroalkyl” refers to C₂-C₈ alkyl in which one to four carbon atomsare independently replaced with a heteroatom from the group consistingof O, S and N.

“Carbocycle” refers to a saturated or unsaturated ring having 3 to 8carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle.Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, arranged as abicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicycle [5,6] or [6,6] system. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl.

A “C₃-C₈ carbocycle” refers to a 3-, 4-, 5-, 6-, 7- or 8-memberedsaturated or unsaturated nonaromatic carbocyclic ring. A C₃-C₈carbocycle group can be unsubstituted or substituted with one or moregroups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),-aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —SR′, —S(O)R′, —S(O)₂R′, —OH, -halogen, —N₃, —NH₂, —NH(R′),—N(R′)₂ and —CN; where each R′ is independently selected from —C₁-C₈alkyl and aryl.

“Alkenyl” refers to an aliphatic hydrocarbon group containing acarbon-carbon double bond which may be straight or branched having 2 to8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl,propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl,hexylenyl, heptenyl, octenyl.

“Alkynyl” refers to an aliphatic hydrocarbon group containing acarbon-carbon triple bond which may be straight or branched having 2 to8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl,propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl,hexylynyl, heptynyl, and octynyl.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—), 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene,propargyl and 4-pentynyl.

“Aryl” or Ar refers to an aromatic or hetero aromatic group, composed ofone or several rings, comprising three to fourteen carbon atoms,preferentially six to ten carbon atoms. The term of “hetero aromaticgroup” refers one or several carbon on aromatic group, preferentiallyone, two, three or four carbon atoms are replaced by O, N, Si, Se, P orS, preferentially by O, S, and N. The term aryl or Ar also refers to anaromatic group, wherein one or several H atoms are replacedindependently by —R′, -halogen, —OR′, or —SR′, —NR′R″, —N═NR′, —N═R′,—NR′R″, —NO₂, —S(O)R′, —S(O)₂R′, —S(O)₂OR′, —OS(O)₂OR′, —PR′R″,—P(O)R′R″, —P(OR′)(OR″), —P(O)(OR′)(OR″) or —OP(O)(OR′)(OR″) wherein R′,R″ are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl,arylalkyl, carbonyl, or pharmaceutical salts.

“Heterocycle” refers to a ring system in which one to four of the ringcarbon atoms are independently replaced with a heteroatom from the groupof O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S.Heterocycles are also described in The Handbook of Chemistry andPhysics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, thedisclosure of which is hereby incorporated by reference. Preferrednonaromatic heterocyclic include, but are not limited to epoxy,aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl,oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl,dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl,pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl,dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl,dihydropyranyl, tetrahydropyridyl, dihydropyridyl,tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as thefused systems resulting from the condensation with a phenyl group.

The term “heteroaryl” or aromatic heterocycles refers to a 5 to 14,preferably 5 to 10 membered aromatic hetero, mono-, bi- or multicyclicring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl,pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl,imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl,1,2,4-thiadiazolyl, isothiazolyl, triazoyl, tetrazolyl, isoquinolyl,benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl,isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting fromthe condensation with a phenyl group.

“Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”,“heterocyclic” and the like refer also to the corresponding “alkylene”,“cycloalkylene”, “alkenylene”, “alkynylene”, “arylene”, “heteroarylene”,“heterocyclene” and the likes which are formed by the removal of twohydrogen atoms.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl and the like.

Examples of a “hydroxyl protecting group” include, but are not limitedto, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranylether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether,triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilylether, triphenylmethylsilyl ether, acetate ester, substituted acetateesters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Such leaving groups are well known in the art,and examples include, but are not limited to, a halide (e.g., chloride,bromide, and iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The following abbreviations may be used herein and have the indicateddefinitions: Boc, tert-butoxy carbonyl; BroP,bromotrispyrrolidinophosphonium hexafluorophosphate; CDI,1,1′-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE,dichloroethane; DCM, dichloromethane; DIAD, diisopropylazodicarboxylate;DIBAL-H, diisobutylaluminium hydride; DIPEA, diisopropylethylamine;DEPC, diethyl phosphorocyanidate; DMA, N,N-dimethyl acetamide; DMAP,4-(N, N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO,dimethylsulfoxide; DTT, dithiothreitol; EDC,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS,electrospray mass spectrometry; HATU, O-(7-azabenzotriazol-1-yl)-N, N,N′, N′-tetramethyluronium hexafluorophosphate; HOBt,1-hydroxybenzotriazole; HPLC, high pressure liquid chromatography; NHS,N-Hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS,phosphate-buffered saline (pH 7.0-7.5); PEG, polyethylene glycol; SEC,size-exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA,trifluoroacetic acid; THF, tetrahydrofuran; Val, valine.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and a disclosed compound.Examples of solvents that form pharmaceutically acceptable solvatesinclude, but are not limited to, water, isopropanol, ethanol, methanol,DMSO, ethyl acetate, acetic acid and ethanolamine.

“Pharmaceutically acceptable excipient” includes any carriers, diluents,adjuvants, or vehicles, such as preserving or antioxidant agents,fillers, disintegrating agents, wetting agents, emulsifying agents,suspending agents, solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions as suitabletherapeutic combinations.

As used herein, “pharmaceutical salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The pharmaceutically acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, such conventional non-toxicsalts include those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, tartaric, citric, methanesulfonic, benzenesulfonic,glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic,fumaric, maleic, lactic and the like. Further addition salts includeammonium salts such as tromethamine, meglumine, epolamine, etc., metalsalts such as sodium, potassium, calcium, zinc or magnesium.

The pharmaceutical salts of the present invention can be synthesizedfrom the parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared viareaction the free acidic or basic forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two. Generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, the disclosure of which is hereby incorporated byreference.

The novel conjugates disclosed herein use the bridge linkers. Examplesof some suitable linkers and their synthesis are shown in FIGS. 1 to 10.

The Bridge Linkers

The synthetic routes to produce bridge linkers as well as thepreparation of the conjugates of drugs to a cell binding molecules ofthe present invention are shown in FIGS. 1-9. The bridge linkers possesstwo elements: a) A Substituent that is acetylenedicarboxyl group thatcan react to a pair of thiols to form covalent thioether bonds, and b) Agroup, such as but not limited to, a disulfide, maleimide, haloacetyl,aldehyde, ketone, azide, amine, alkoxyamine and hydrazide, capable ofreaction with a drug. The bridge substituents of acetylenedicarboxyl canbe introduced by direct condensation of acetylenedicarboxylic acid withan amine, an alcohol, or a thiol group to form amide, ester or thioesterbonds at both the ends of acetylenedicarboxyl sites. The synthesis ofthese bridge linkers is exampled in the FIGS. 1, 3, 4, 5, 6, 7, 8 and 9.The bridge substituents of acetylenedicarboxyl can be introduced bycondensation of acetylene with acid halides or acid anhydrides to formcarbon-carbon bonds at both the ends of acetylenedicarboxyl sites. Thesynthesis of these bridge linkers is exampled in the FIGS. 2 and 10.

Preferably, the bridge linkers are compounds of the Formula (I) below:

Wherein the acetylenedicarboxyl group on the linker is capable ofreacting with a pair of sulfur atoms of the cell-binding agent; Thesulfur atoms are preferred pairs of thiols reduced from the interchaindisulfide bonds of the cell-binding agent by a reducing agent, such asDTT and/or TCEP;

Z₁ and Z₂ are the same or different a function group that enables toreact with a cytotoxic drug, to form a disulfide, thioether, thioester,peptide, hydrazone, ether, ester, carbamate, carbonate, amine(secondary, tertiary, or quarter), imine, cycloheteroalkyane,heteroaromatic, alkoxime or amide bond;

R₁ and R₂ are the same or different, and are absent, linear alkyl havingfrom 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6carbon atoms, linear, branched or cyclic alkenyl or alkynyl, or 1˜6carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula(OCH₂CH₂)_(p), or polypropyleneoxy unit of formula (OCH₂(CH₃)CH₂)_(p)wherein p is an integer from 0 to about 1000, or combination thereof.

Additionally R₁ and R₂ are respectively a chain of atoms selected fromC, N, O, S, Si, and P, preferably having 0˜500 atoms, which covalentlyconnects to X₁ or X₂ and Z₁ or Z₂. The atoms used in forming the R₁ andR₂ may be combined in all chemically relevant ways, such as formingalkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters,amines, imines, polyamines, hydrazines, hydrazones, amides, ureas,semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes,amino acids, peptides, acyloxylamines, hydroxamic acids, or combinationthereof.

X₁ and X₂ are independently selected from N(R₃), O, S or CH₂; Wherein R₃is H, linear alkyl having from 1-6 carbon atoms, branched or cyclicalkyl having from 3 to 6 carbon atoms, linear, branched or cyclicalkenyl or alkynyl, or 1˜6 carbon atoms of esters, ether, amide, orpolyethyleneoxy unit of formula (OCH₂CH₂)_(p), wherein p is an integerfrom 0 to about 1000, or combination thereof.

In another embodiment, R₁, R₂, and R₃, can be respectively a chain ofatoms selected from C, N, O, S, Si, and P which covalently connects thecell-surface binding molecule and/or the conjugated drug. The atoms usedin forming the bridge linker may be combined in all chemically relevantways, such as forming alkylene, alkenylene, and alkynylene, ethers,polyoxyalkylene, esters, amines, imines, polyamines, hydrazines,hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines,alkoxylamines, urethanes, amino acids, acyloxylamines, hydroxamic acids,and many others. In addition, it is to be understood that the atomsforming the linker (L) may be either saturated or unsaturated, or may beradicals, or may be cyclized upon each other to form divalent cyclicstructures, including cyclo alkanes, cyclic ethers, cyclic amines,arylenes, heteroarylenes, and the like in the linker.

Examples of the functional groups, Z₁ and Z₂, which enable linkage of acytotoxic drug, include groups that enable linkage via a disulfide,thioether, thioester, peptide, hydrazone, ester, carbamate, carbonate,alkoxime or an amide bond. Such functional groups include, but are notlimited to, thiol, disulfide, amino, carboxy, aldehydes, ketone,maleimido, haloacetyl, hydrazines, alkoxyamino, and/or hydroxy.

Examples of the functional groups, Z₁ and Z₂, that enable reaction withthe terminal of amine of a drug/cytotoxic agent can be, but not limitedto, N-hydroxysuccinimide esters, p-nitrophenyl esters, dinitrophenylesters, pentafluorophenyl esters, carboxylic acid chlorides orcarboxylic acid anhydride; With the terminal of thiol can be, as but notlimited to, pyridyldisulfides, nitropyridyldi sulfides, maleimides,haloacetates, methylsulfone phenyloxadiazole (ODA), carboxylic acidchlorides and carboxylic acid anhydride; With the terminal of ketone oraldehyde can be, as but not limited to, amines, alkoxyamines,hydrazines, acyloxylamine, or hydrazide; With the terminal of azide canbe, as but not limited to, alkyne. Examples of these function groups aredisplayed below:

Wherein X₁ is F, Cl, Br, I or Lv; X₂ is O, NH, N(R₁), or CH₂; R₅ and R₃are H, R₁, aromatic, heteroaromatic, or aromatic group wherein one orseveral H atoms are replaced independently by —R₁, -halogen, —OR₁, —SR₁,—NR₁R₂, —NO₂, —S(O)R₁, —S(O)₂R₁, or —COOR₁; Lv is a leaving groupselected from nitrophenol; N-hydroxysuccinimide (NHS); phenol;dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol;monofluorophenol; pentachlorophenol; triflate; imidazole;dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate;mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrides formedits self, or formed with the other anhydride, e.g. acetyl anhydride,formyl anhydride; or a intermediate molecule generated with acondensation reagent for peptide coupling reactions, or for Mitsunobureactions.

In preferred embodiments, R₁, R₂, and R₃, are linear alkyl having from1-6 carbon atoms, or polyethyleneoxy unit of formula (OCH₂CH₂)_(p),p=1˜100.

The key step of synthesis of the bridge linker containingacetylenedicarboxyl groups is the condensation of theacetylenedicarboxylic acid, or its acid derivatives, with the othercomponents containing an amine (1° or 2° amines), alcohol, or thiol ontheir terminal, as shown in the following scheme (Ia):

Wherein X is X₁ or X₂ described in Formula (I) as NH, N(R₃), O, or S; Ris R₁ and/or R₂ that described in Formula (I); R₃ is the same defined inFormula (I).

Lv₁ and Lv₂ are the same or independently OH; F; Cl; Br; I; nitrophenol;N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol;tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol;triflate; imidazole; dichlorophenol; tetrachlorophenol;1-hydroxybenzotriazole; tosylate; mesylate;2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrides formed its self, orformed with the other anhydride, e.g. acetyl anhydride, formylanhydride; or a intermediate molecule generated with a condensationreagent for peptide coupling reactions, or for Mitsunobu reactions, e.g.condensation reagents are: EDC(N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), DCC(Dicyclohexyl-carbodiimide), N,N′-Diisopropylcarbodiimide (DIC),N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate(CMC, or CME-CDI), 1,1′-Carbonyldiimidazole (CDI), TBTU(O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate),N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU),(Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate(BOP), (Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), Diethyl cyanophosphonate (DEPC),Chloro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate,1-[Bis(dimethylamino)methylene]-1H1,2,3-triazolo[4, 5-b]pyridinium3-oxid hexafluorophosphate (HATU), 1-[(Dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium 3-oxidehexafluorophosphate (HDMA), 2-Chloro-1,3-dimethylimidazolidiniumhexafluorophosphate (CIP), Chlorotripyrrolidinophosphoniumhexafluorophosphate (PyCloP),Fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate(BTFFH), N,N,N′,N′-Tetramethyl-S-(1-oxido-2-pyridyl)thiuroniumhexafluorophosphate, O-(2-Oxo-1(2H)pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TPTU),S-(1-Oxido-2-pyridyl)-N,N,N′,N′-tetramethylthiuronium tetrafluoroborate,O-[(Ethoxycarbonyl) cyanomethylenamino]-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HOTU), (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluoro-phosphate(COMU),O-(Benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene) uroniumhexafluorophosphate (HBPyU), N-Benzyl-N′-cyclohexylcarbodiimide (with,or without polymer-bound), Dipyrrolidino(N-succinimidyloxy)carbeniumhexafluoro-phosphate (HSPyU), Chlorodipyrrolidinocarbeniumhexafluorophosphate (PyClU), 2-Chloro-1,3-dimethylimidazolidiniumtetrafluoroborate(CIB), (Benzotriazol-1-yloxy) dipiperidinocarbeniumhexafluorophosphate (HBPipU),O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TCTU), Bromotris(dimethylamino)-phosphoniumhexafluorophosphate (BroP), Propylphosphonic anhydride (PPACA, T3P®),2-Morpholinoethyl isocyanide (MEI),N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium hexafluorophosphate(HSTU), 2-Bromo-1-ethyl-pyridinium tetrafluoroborate (BEP),O-[(Ethoxycarbonyl)cyanomethylenamino]-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TOTU),4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(MMTM, DMTMM), N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uroniumtetrafluoroborate (TSTU),O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TDBTU), 1,1′-(Azodicarbonyl)dipiperidine (ADD),Di-(4-chlorobenzyl) azodicarboxylate (DCAD), Di-tert-butylazodicarboxylate (DBAD), Diisopropyl azodicarboxylate (DIAD), Diethylazodicarboxylate (DEAD).

When X is CH₂, wherein the acetylenedicarboxyl group on the bridgelinker connects to the other components of linker through C—C bonds,then the key step of synthesis of the bridge linker containingacetylenedicarboxyl groups is the condensation ofbis(trimethylsilyl)acetylene, or acetylene bis-magnesiums (Grignardreagent), or acetylene bis-lithiums (dilithioacetylene), or otherdi-metal acetylide with acid halides or acid anhydrides, depicted asfollowing reaction equations (Ib), (Ic), (Id), (Ie), (If), (Ig) and(Ih):

Wherein M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.

The detail examples of the synthesis of the bridge linkers are shown inthe FIGS. 1˜10. Normally the bridge substituents of acetylenedicarboxylcan be condensated with linker components containing function groupscapable to react to drugs of desired conjugation.

Cell-Binding Agent-Drug Conjugates

The conjugates of the present invention can be represented by thefollowing formula,

wherein Cb is a cell-binding agent, L is the acetylenedicarboxyl bridgelinker, Drug₁ and Drug₂ are a drug molecule, n is an integer from 1 to20, and two S (sulfur) elements from Cb bridgely link to L, whichcovalently connects two or more drugs (per bridge linker L).

The bridge linker L may be composed of one or more linker components.Exemplary linker components include 6-maleimidocaproyl (“MC”),maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”),alanine-phenylalanine (“ala-phe” or “af”), p-aminobenzyloxycarbonyl(“PAB”), 4-thiopentanoate (“SPP”), 4-(N-maleimidomethyl)cyclohexane-1carboxylate (“MCC”), (4-acetyl)aminobenzoate (“SIAB”), 4-thio-butyrate(SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), ethyleneoxy—CH₂CH₂O— as one or more repeating units (“EO” or “PEO”). Additionallinker components are known in the art and some are described herein.

Example structures of these components containing linkers are:

Preferably, the conjugates have the following Formula (II):

wherein:

Cb represents a cell-binding agent, preferably an antibody, whichconjugates to Drug₁ and Drug₂ via a pair of sulfur atoms (thiols). Theconjugatable thiol groups can generally be generated from TCEP or DTTreduction of pairs of disulfide bonds on the surface of cell-bindingmolecule.

Drug₁ and Drug₂ represent the same or different cytotoxic agents, linkedto the cell-binding agent via the bridge linker through an alkyl,alkylene, alkenylene, alkynylene, ether, polyoxyalkylene, ester, amine,imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide,carbazide, alkoxyamine, urethanes, amino acid, peptide, acyloxylamine,hydroxamic acid, disulfide, thioether, thioester, carbamate, carbonate,heterocyclic ring, heteroalkyl, heteroaromatic, or alkoxime bond, orcombination thereof.

n is 1˜20; R₁, R₂, X₁ and X₂ are described the same previously inFormula (I).

As described in more detail below, Drug₁ and Drug₂ can be any of manysmall molecule drugs, including, but not limited to, tubulysins,calicheamicins, auristatins, maytansinoids, CC-1065 analogs, morpholinosdoxorubicins, taxanes, cryptophycins, epothilones, and benzodiazepinedimers (e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin),indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidinobenzodiazepines).

To synthesize the conjugate, the cell-binding agent can be firstmodified with the bridge linkers of the present invention throughreduction of disulfide bonds of the cell-binding molecule. The yielded apair of free thiols can react to the bridge linker of Formula (I) at pH5˜9 aqueous media with or without addition of 0˜30% of water mixable(miscible) organic solvents, such as DMA, DMF, ethanol, methanol,acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, orethylene diol, to introduce the reactive groups of Z₁ and Z₂ containingdisulfide, maleimido, haloacetyl, azide, 1-yne, ketone, aldehyde,alkoxyamino, or hydrazide groups. Then a reactive group of a cytotoxicagent reacts to the modified cell-binding molecule accordingly. Forexample, synthesis of the cell-binding agent-drug conjugates linked viadisulfide bonds is achieved by a disulfide exchange between thedisulfide bond in the modified cell-binding agent and a drug containinga free thiol group. Synthesis of the cell-binding agent-drug conjugateslinked via thioether is achieved by reaction of the maleimido orhaloacetyl or ethylsulfonyl modified cell-binding agent and a drugcontaining a free thiol group. Synthesis of conjugates bearing an acidlabile hydrazone can be achieved by reaction of a carbonyl group withthe hydrazide moiety in the linker, by methods known in the art (see,for example, P. Hamann et al., Hinman, L. M., et al, Cancer Res. 53,3336-334, 1993; B. Laguzza et al., J. Med. Chem., 32; 548-555, 1959; P.Trail et al., Cancer Res., 57; 100-105, 1997). Synthesis of conjugatesbearing triazole linkage can be achieved by reaction of a 1-yne group ofthe drug with the azido moiety in the linker, through the clickchemistry (Huisgen cycloaddition) (Lutz, J-F. et al, 2008, Adv. DrugDel. Rev. 60, 958-970; Sletten, E. M. et al 2011, Acc Chem. Research 44,666-676).

Alternatively, the drug can react with the bridge linkers of the presentinvention that have conjugated to a cell-binding molecule to give amodified cell-binding molecule linker of Formula (III) bearingfunctionalities. For example, a thiol-containing drug can be reachedwith the modified cell-binding molecule bridge linker of Formula (III)bearing a maleimdo, or a haloacetyl, or an ethylsulfonyl substituent atpH 5.5˜9.0 in aqueous buffer to give a cell-binding molecule-drugconjugate via a thioether linkage. A thiol-containing drug can undergodisulfide exchange with a modified bridge linker of Formula (III)bearing a pyridyldithio moiety to give a conjugate a disulfide bondlinkage. A drug bearing a hydroxyl group or a thiol group can be reactedwith a modified bridge linker of Formula (III) bearing a halogen,particularly the alpha halide of carboxylates, in the presence of a mildbase, e.g. pH 8.0˜9.5, to give a modified drug bearing an ether or thiolether link. A hydroxyl group containing drug can be condensed with abridge cross linker of Formula (I) bearing a carboxyl group, in thepresence of a dehydrating agent, such as EDC or DCC, to give esterlinkage, then the subject drug modified bridge linker undergoes theconjugation with a cell-binding molecule. A drug containing an aminogroup can condensate with a carboxyl ester of NHS, imidazole,nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol;pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol;pentachlorophenol; triflate; imidazole; dichlorophenol;tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate;2-ethyl-5-phenylisoxazolium-3′-sulfonate on the cell-bindingmolecule-bridge linker of Formula (III) to give a conjugate via amidebond linkage.

The conjugate may be purified by standard biochemical means, such as gelfiltration on a Sephadex G25 or Sephacryl S300 column, adsorptionchromatography, and ion exchange or by dialysis. In some cases, a smallmolecule as a cell-binding agent (e.g. folic acid, melanocytestimulating hormone, EGF etc) conjugated with a small molecular drugscan be purified by chromatography such as by HPLC, medium pressurecolumn chromatography or ion exchange chromatography.

Modified Cell-Binding Agents/Molecules

The cell-binding agent modified by reaction with linkers of the presentinvention are preferably represented by the Formula (III)

Wherein Cb, Z₁, Z₂, n, R₁, R₂, X₁, and X₂ are defined the same as inFormula (I) and (II).

In preferred embodiments, Z₁ and Z₂ are a disulfide substituent,maleimido, haloacetyl, alkoxyamine, azido, ketone, aldehyde, hydrazine,alkyne, an N-hydroxysuccinimide ester, or a carboxyl ester formed withphenol; dinitrophenol; pentafluorophenol; tetrafluorophenol;difluorophenol; monofluorophenol; pentachlorophenol; triflate;imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole;tosylate; mesylate; 2-ethyl-5-phenylisoxa-zolium-3′-sulfonate. Z₁ and Z₂can then react with a cytotoxic agent through disulfide, thioether,hydrazone, amide, alkoxime, carbamate, ester, ether bond orheteroaromatic ring. The modified cell-binding agent can be prepared viaa reaction of the cell-binding agent with the bridge linkers of Formula(I) as described in Formula (II) above.

In order to achieve a higher conjugation yield of the alkyne group onthe bridge linkers with a pair of free thiols on the cell-bindingmolecule, preferably on an antibody, a small percentage of organicco-solvent may be required to add to the reaction mixture, as well inthe solution after the reaction to maintain solubility of the Formula(III) in aqueous solution. To modify the cell-binding agents, thecross-linking reagent (bridge linker) of Formula (I) can be firstdissolved in a polar organic solvent that is miscible with water, forexample different alcohols, such as methanol, ethanol, and propanol,acetone, acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethylformamide (DMF), dimethyl acetamide (DMA), or dimethylsulfoxide (DMSO)at a high concentration, for example 1-500 mM. Meanwhile, thecell-binding molecule, such as antibody dissolved in an aqueous bufferpH 5˜9.5, preferably pH 6˜8.5, at 1˜35 mg/ml concentration was treatedwith 1-20 equivalent of TCEP or DTT for 20 min to 12 hour. After thereduction, DTT can be removed by SEC chromatographic purification. TCEPcan be optionally removed by SEC chromatography too, or staying in thereaction mixture for the next step reaction without purification.Furthermore, the reduction of antibodies or the other cell-bindingagents with TCEP can be performed with a bridge linker of Formula (I),for which the cross-linking conjugation for the cell-binding moleculescan be achieved simultaneously along with the TCEP reduction.

The aqueous solutions for the modification of cell-binding agents arebuffered between pH 6 and 9, preferably between 6.5 and 7.5 and cancontain any non-nucleophilic buffer salts useful for these pH ranges.Typical buffers include phosphate, triethanolamine HCl, HEPES, and MOPSbuffers, which can contain additional components, such as cyclodextrins,sucrose and salts, for examples, NaCl and KCl. After the addition of thebridge linker of Formula (I) into the solution containing the reducedcell-binding molecules, the reaction mixture is incubated at atemperature of from 4° C. to 45° C., preferably at ambient temperature.The progress of the reaction can be monitored by measuring the decreasein the absorption at 254 nm, or increase in the absorption at 280 nm, orthe other appropriate wavelength. After the reaction is complete,isolation of the modified cell-binding agent can be performed in aroutine way, using for example gel filtration chromatography, oradsorptive chromatography.

The extent of modification can be assessed by measuring the absorbanceof the nitropyridine thione, dinitropyridine dithione, pyridine thione,carboxamidopyridine dithione and dicarboxamidopyridine dithione groupreleased via UV spectra. For the conjugation without a chromophoregroup, the modification or conjugation reaction can be monitored byLC-MS, preferably by UPLC-QTOF mass spectrometry, or Capillaryelectrophoresis-mass spectrometry (CE-MS). The bridge cross-linkersdescribed herein have diverse functional groups that can react with anydrugs, preferably cytotoxic agents that possess a suitable substituent.For examples, the modified cell-binding molecules bearing an amino orhydroxyl substituent can react with drugs bearing anN-hydroxysuccinimide (NHS) ester, the modified cell-binding moleculesbearing a thiol substituent can react with drugs bearing a maleimido orhaloacetyl group. Additionally, the modified cell-binding moleculesbearing a carbonyl (ketone or aldehyde) substituent can react with drugsbearing a hydrazide or an alkoxyamine. One skilled in the art canreadily determine which linker to use based on the known reactivity ofthe available functional group on the linkers.

Modified Cytotoxic Drugs

The cytotoxic drugs modified by reaction with cross-linkers of thepresent invention are preferably represented by the Formula (IV):

Wherein Drug₁, Drug₂, Z₁, Z₂, n, R₁, R₂, X₁, and X₂ are defined the sameas in Formula (I) and (II).

The modified drugs can be prepared via reaction of the drug with thelinkers of the Formula (I) to give a modified drug of Formula (IV)bearing functionality of an acetylene-di-carboxyl group capable ofreacting with a pair of thiol groups of a cell-binding agent. Theacetylenedicarboxyl group is synthesized through condensation withacetylene via the methods described in reaction equation (Ia), (Ib),(Ic), (Id), (Ie), (If), (Ig) and (Ih). But for drugs containing a thiol,or the drugs undergoing to link a cell-binding molecule via the bridgelinkers through thioether, thioester or disulfide bond, it is thereforepreferred that the Drug₁ or Drug₂ may be synthesized to connect to R₁,or R₂ in a piece of components via the linkage of thioether, thioesteror disulfide bond first. Then the synthesized R₁-Drug₁ or R₂-Drug₂component is assembled to an acetylenedicarboxyl group to form thebridge linker modified drugs of Formula (IV).

For examples of the synthesis, a thiol-containing drug can be reactedwith the linker of components R₁ or R₂ bearing a maleimdo substituent atneutral pH in aqueous buffer to give a R₁-Drug₁ or R₂-Drug₂ compartmentbearing thioether linkage, and following by condensation with acompartment of acetylenedicarboxyl group to give a modified drug ofFormula (IV) bearing thioether linkage. A drug bearing a hydroxyl groupcan be reacted with a linker component R₁ or R₂ bearing a halogen, or atosylate, or a mesylate, in the presence of a mild base, to give aR₁-Drug₁ or R₂-Drug₂ compartment bearing ether linkage, and following bycondensation with a compartment of acetylenedicarboxyl group to give amodified drug of Formula (IV) bearing thioether linkage. A hydroxylgroup containing drug can be condensed with a linker of Formula (I)bearing a carboxyl group, in the presence of a dehydrating agent, suchas EDC or dicyclohexylcarbodiimide (DCC), to give a modified drug ofFormula (IV) via ester linkage. A drug bearing a thiol group can alsoreact the linker of components R₁ or R₂ bearing a maleimido or avinylsulfonyl, or a haloacetyl group, give a R₁-Drug₁ or R₂-Drug₂compartment bearing thioether linkage, and following by condensationwith a compartment of acetylenedicarboxyl group to give a modified drugof Formula (IV) bearing thioether linkage. An amino group containingdrug can similarly undergo condensation with a carboxyl group on thebridge linker of Formula (I) to give a modified drug of Formula (IV)bearing amide bonds. The modified drug can be purified by standardmethods such as column chromatography over silica gel or alumina,crystallization, preparatory thin layer chromatography, ion exchangechromatography, or HPLC.

Cell-Binding Agents

The cell-binding molecule that comprises the conjugates and the modifiedcell-binding agents of the present invention may be of any kindpresently known, or that become known, molecule that binds to, complexeswith, or reacts with a moiety of a cell population sought to betherapeutically or otherwise biologically modified.

The cell binding agents include, but are not limited to, large molecularweight proteins such as, for example, full-length antibodies (polyclonalantibodies, monoclonal antibodies, dimers, multimers, multispecificantibodies (e.g., bispecific antibodies); single chain antibodies;fragments of antibodies such as Fab, Fab′, F(ab′)₂, F, [Parham, J.Immunol. 131, 2895-2902 (1983)], fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR's, diabody, triabody,and epitope-binding fragments of any of the above whichimmuno-specifically bind to cancer cell antigens, viral antigens,microbial antigens or a protein generated by the immune system that iscapable of recognizing, binding to a specific antigen or exhibiting thedesired biological activity (Miller et al (2003) J. of Immunology170:4854-4861); interferons (such as type I, II, III); peptides;lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF,interferon-gamma (IFN-γ); hormones such as insulin, TRH (thyrotropinreleasing hormones), MSH (melanocyte-stimulating hormone), steroidhormones, such as androgens and estrogens, melanocyte-stimulatinghormone (MSH); growth factors and colony-stimulating factors such asepidermal growth factors (EGF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), transforming growth factors (TGF),such as TGFα, TGFβ, insulin and insulin like growth factors (IGF-I,IGF-II) G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today, 5, 155-158(1984)]; vaccinia growth factors (VGF); fibroblast growth factors(FGFs); smaller molecular weight proteins, poly-peptide, peptides andpeptide hormones, such as bombesin, gastrin, gastrin-releasing peptide;platelet-derived growth factors; interleukin and cytokines, such asinterleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factors,granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins,such as folate; apoproteins and glycoproteins, such as transferrin[O'Keefe et al, 260 J. Biol. Chem. 932-937 (1985)]; sugar-bindingproteins or lipoproteins, such as lectins; cell nutrient-transportmolecules; and small molecular inhibitors, such as prostate-specificmembrane antigen (PSMA) inhibitors and small molecular tyrosine kinaseinhibitors (TKI), non-peptides or any other cell binding molecule orsubstance, such as bioactive polymers (Dhar, et al, Proc. Natl. Acad.Sci. 2008, 105, 17356-61); bioactive dendrimers (Lee, et al, Nat.Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci.2009, 106, 685-90); nanoparticles (Liong, et al, ACS Nano, 2008, 19,1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et al,Bioconjugate Chem. 2008, 19, 1309-12); liposomes (Medinai, et al, Curr.Phar. Des. 2004, 10, 2981-9); viral capsides (Flenniken, et al, VirusesNanotechnol. 2009, 327, 71-93).

In general, a monoclonal antibody is preferred as a cell-surface bindingagent if an appropriate one is available. And the antibody may bemurine, human, humanized, chimeric, or derived from other species.

Production of antibodies used in the present invention involves in vivoor in vitro procedures or combinations thereof. Methods for producingpolyclonal anti-receptor peptide antibodies are well-known in the art,such as in U.S. Pat. No. 4,493,795 (to Nestor et al). A monoclonalantibody is typically made by fusing myeloma cells with the spleen cellsfrom a mouse that has been immunized with the desired antigen (Köhler,G.; Milstein, C. (1975). Nature 256: 495-497). The detailed proceduresare described in “Antibodies—A Laboratory Manual”, Harlow and Lane,eds., Cold Spring Harbor Laboratory Press, New York (1988), which isincorporated herein by reference. Particularly monoclonal antibodies areproduced by immunizing mice, rats, hamsters or any other mammal with theantigen of interest such as the intact target cell, antigens isolatedfrom the target cell, whole virus, attenuated whole virus, and viralproteins. Splenocytes are typically fused with myeloma cells usingpolyethylene glycol (PEG) 6000. Fused hybrids are selected by theirsensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomasproducing a monoclonal antibody useful in practicing this invention areidentified by their ability to immunoreact specified receptors orinhibit receptor activity on target cells.

A monoclonal antibody used in the present invention can be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques, such as using protein-A affinitychromatography; anion, cation, hydrophobic, or size exclusivechromatographies (particularly by affinity for the specific antigenafter protein A, and sizing column chromatography); centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media. An exemplary synthetic medium is Dulbecco's minimalessential medium (DMEM; Dulbecco et al., Virol. 8, 396 (1959))supplemented with 4.5 gm/l glucose, 0˜20 mM glutamine, 0˜20% fetal calfserum, several ppm amount of heavy metals, such as Cu, Mn, Fe, or Zn,etc, or/and the heavy metals added in their salt forms, and with ananti-foaming agent, such as polyoxyethylene-polyoxypropylene blockcopolymer.

In addition, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with an oncovirus, suchas Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4)) orKaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also beproduced via an anti-receptor peptide or peptides containing thecarboxyl terminal as described well-known in the art. See Niman et al.,Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et al., Proc.Natl. Acad. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry34(20): 6675-6688, (1995). Typically, the anti-receptor peptide or apeptide analog is used either alone or conjugated to an immunogeniccarrier, as the immunogen for producing anti-receptor peptide monoclonalantibodies.

There are also a number of other well-known techniques for makingmonoclonal antibodies as binding molecules in this invention.Particularly useful are methods of making fully human antibodies. Onemethod is phage display technology which can be used to select a rangeof human antibodies binding specifically to the antigen using methods ofaffinity enrichment. Phage display has been thoroughly described in theliterature and the construction and screening of phage display librariesare well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13(1994); Little et al, Biotechnol Adv. 12(3):539-55 (1994); Clackson etal., Nature 352:264-628 (1991); Huse et al., Science 246:1275-1281(1989).

Monoclonal antibodies derived by hybridoma technique from anotherspecies than human, such as mouse, can be humanized to avoid humananti-mouse antibodies when infused into humans. Among the more commonmethods of humanization of antibodies are complementarity-determiningregion grafting and resurfacing. These methods have been extensivelydescribed, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al,Immunol Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 13: 1619-33(2008); Lazar et al, Mol Immunol. 44(8):1986-98 (2007); Li et al, Proc.Natl. Acad. Sci. USA. 103(10):3557-62 (2006) each incorporated herein byreference. Fully human antibodies can also be prepared by immunizingtransgenic mice, rabbits, monkeys, or other mammals, carrying largeportions of the human immunoglobulin heavy and light chains, with animmunogen. Examples of such mice are: the Xenomouse. (Abgenix/Amgen),the HuMAb-Mouse (Medarex/BMS), the VelociMouse (Regeneron), see alsoU.S. Pat. Nos. 6,596,541, 6,207,418, U.S. Pat. No. 6,150,584, U.S. Pat.No. 6,111,166, U.S. Pat. No. 6,075,181, U.S. Pat. No. 5,922,545, U.S.Pat. Nos. 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In humantherapy, murine variable regions and human constant regions can also befused to construct called “chimeric antibodies” that are considerablyless immunogenic in man than murine mAbs (Kipriyanov et al, MolBiotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9(2002) each incorporated herein by reference). In addition,site-directed mutagenesis in the variable region of an antibody canresult in an antibody with higher affinity and specificity for itsantigen (Brannigan et al, Nat Rev Mol Cell Biol. 3:964-70, (2002));Adams et al, J Immunol Methods. 231:249-60 (1999)) and exchangingconstant regions of a mAb can improve its ability to mediate effectorfunctions of binding and cytotoxicity.

Antibodies immunospecific for a malignant cell antigen can also beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., chemical synthesis or recombinant expressiontechniques. The nucleotide sequence encoding antibodies immunospecificfor a malignant cell antigen can be obtained commercially, e.g., fromthe GenBank database or a database like it, the literature publications,or by routine cloning and sequencing.

Apart from an antibody, a peptide or protein that bind/block/target orin some other way interact with the epitopes or corresponding receptorson a targeted cell can be used as a binding molecule. These peptides orproteins could be any random peptide or proteins that have an affinityfor the epitopes or corresponding receptors and they don't necessarilyhave to be of the immunoglobulin family. These peptides can be isolatedby similar techniques as for phage display antibodies (Szardenings, JRecept Signal Transduct Res. 2003; 23(4):307-49). The use of peptidesfrom such random peptide libraries can be similar to antibodies andantibody fragments. The binding molecules of peptides or proteins may beconjugated on or linked to a large molecules or materials, such as, butis not limited, an albumin, a polymer, a liposome, a nano particle, adendrimer, as long as such attachment permits the peptide or protein toretain its antigen binding specificity.

Examples of antibodies used for conjugation of drugs via the bridgelinkers of this prevention for treating cancer, autoimmune disease,and/or infectious disease include, but are not limited to, 3F8(anti-GD2), Abagovomab (anti CA-125), Abciximab (anti CD41 (integrinalpha-IIb), Adalimumab (anti-TNF-α), Adecatumumab (anti-EpCAM, CD326),Afelimomab (anti-TNF-α); Afutuzumab (anti-CD20), Alacizumab pegol(anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab (Campath, MabCampath,anti-CD52), Altumomab (anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab(IMA-638, anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA),Aselizumab (anti-L-selectin (CD62L), Atlizumab (tocilizumab, Actemra,RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor),Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, antiCD25 (αchain of IL-2 receptor), Bavituximab (anti-phosphatidylserine),Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B,anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCL11(eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen),Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrinII beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE,anti-CD19), Brentuximab (cAC10, anti-CD30 TNFRSF8), Briakinumab(anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242,anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49(anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimziaanti-TNF-α), Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumabbogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4),Clivatuzumab (anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261(anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab(Zenapax, anti-CD25 (α chain of IL-2 receptor)), Daratumumab (anti-CD38(cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab(anti-B-lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab(anti-endotoxin), Edrecolomab (Panorex, MAb17-1A, anti-EpCAM),Efalizumab (Raptiva, anti-LFA-1 (CD11a), Efungumab (Mycograb,anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6),Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin),Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab(Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrinα_(v)β₃), Exbivirumab (anti-hepatitis B surface antigen), Fanolesomab(NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor),Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratorysyncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virusglycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80),Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD147 (basigin)),Gemtuzumab (anti-CD33), Girentuximab (anticarbonic anhydrase 9),Glembatumumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-α),Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4),Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125),Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade,anti-TNF-α), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (a chain ofIL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152),Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab(CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab(anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2),Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surfaceantigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab(anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Maslimomab(anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria,anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74),Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside),Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratorysyncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab(anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrinα₄), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab(anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab,Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)),Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R α), Omalizumab(Xolair, anti-IgE Fc region), Oportuzumab (anti-EpCAM), Oregovomab(OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab(anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis,anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF,anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa), Pascolizumab(anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab (Omnitarg,2C4, anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab(anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab(anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10,anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)),Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2),Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin,protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B),Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera,Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab(anti-IFN-α), Rovelizumab (LeukArrest, anti-CD11, CD18), Ruplizumab(Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab(anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab(anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart)MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab(anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab(anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyteantigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab(anti-integrin α_(IIb)β₃), Talizumab (anti-IgE), Tanezumab (anti-NGF),Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factorA), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40),Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab,(anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13),Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor),Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab(Herceptin, (anti-HER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumabcelmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus),Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12,IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab, (anti-integrinα₄β₇), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1),Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),Volociximab (anti-integrin α₅β₁), Votumumab (HumaSPECT, anti-tumorantigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab(HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab(anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept(Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron RegulatoryProtein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst. formelanoma and solid tumors), J591 (anti-PSMA, Weill Cornell MedicalSchool for prostate cancers), 225.28S [anti-HMW-MAA (High molecularweight-melanoma-associated antigen), Sorin Radiofarmaci S.R.L. (Milan,Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst.USA for colorectal and gastric cancers), CYT-356 (Oncoltad®, forprostate cancers), HNK20 (OraVax Inc. for respiratory syncytial virus),ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, PeregrinePharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA,TNF-alpha; TNFSF2), from Abbott/Knoll for Sepsis toxic shock], MEDI-500[T10B9, anti-CD3, TRαβ (T cell receptor alpha/beta), complex, fromMedImmune Inc for Graft-versus-host disease], RING SCAN [anti-TAG 72(tumour associated glycoprotein 72), from Neoprobe Corp. for Breast,Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial celladhesion molecule), anti-TACSTD1 (Tumor-associated calcium signaltransducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen;M4S; tumor antigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian,Prostate cancers and NHL]; LymphoCide (Immunomedics, NJ), Smart ID10(Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune(BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics,NJ), IMC-1C11 (ImClone Systems, NJ) and Cetuximab (ImClone, NJ).

Other antibodies as cell binding molecules/ligands include, but are notlimited to, are antibodies against the following antigens:Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various cancers),CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6(carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alphafetoprotein (carcinomas), CA242 (colorectal), placental alkalinephosphatase (carcinomas), prostate specific antigen (prostate),prostatic acid phosphatase (prostate), epidermal growth factor(carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma),CD3 epsilon (T cell lymphoma, lung, breast, gastric, ovarian cancers,autoimmune diseases, malignant ascites), CD19 (B cell malignancies),CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiplemyeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmunediseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma,leukemia (CLL)), CD51 (Metastatic melanoma, sarcoma), CD52 (leukemia),CD56 (small cell lung cancers, ovarian cancer, Merkel cell carcinoma,and the liquid tumor, multiple myeloma), CD66e (cancers), CD70(metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74(multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin (carcinomas),CD221 (solid tumors), CD227 (breast, ovarian cancers), CD262 (NSCLC andother cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3(colorectal, gastric cancers), CEACAM5 (carcinoembryonic antigen; CEA,CD66e) (breast, colorectal and lung cancers), DLL4 (delta-like-4), EGFR(Epidermal Growth Factor Receptor, various cancers), CTLA4 (melanoma),CXCR4 (CD184, Heme-oncology, solid tumors), Endoglin (CD105, solidtumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck,colon, NHL prostate, and ovarian cancers), ERBB2 (Epidermal GrowthFactor Receptor 2; lung, breast, prostate cancers), FCGR1 (autoimmunediseases), FOLR (folate receptor, ovarian cancers), GD2 ganglioside(cancers), G-28 (a cell surface antigen glyvolipid, melanoma), GD3idiotype (cancers), Heat shock proteins (cancers), HER1 (lung, stomachcancers), HER2 (breast, lung and ovarian cancers), HLA-DR10 (NHL),HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin(carcinoma), IGF1R (insulin-like growth factor 1 receptor, solid tumors,blood cancers), IL-2 receptor (interleukin 2 receptor, T-cell leukemiaand lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, RA,Castleman's disease, IL6 dependent tumors), Integrins (αvβ3, α5β1, α6β4,αllβ3, α5β5, αvβ5, for various cancers), MAGE-1 (carcinomas), MAGE-2(carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrinreceptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning4-domains subfamily A member 1, Non-Hodgkin's B cell lymphoma,leukemia), MUC1 or MUC1-KLH (breast, ovarian, cervix, bronchus andgastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA(colorectal), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1(membrane-spanning 4-domains subfamily A, small cell lung cancers, NHL),Nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas), Paratopeof anti-(N-glycolylneuraminic acid, Breast, Melanoma cancers), PLAP-liketesticular alkaline phosphatase (ovarian, testicular cancers), PSMA(prostate tumors), PSA (prostate), ROBO4, TAG 72 (tumour associatedglycoprotein 72, AML, gastric, colorectal, ovarian cancers), T celltransmembrane protein (cancers), Tie (CD202b), TNFRSF10B (tumor necrosisfactor receptor superfamily member 10B, cancers), TNFRSF13B (tumornecrosis factor receptor superfamily member 13B, multiple myeloma, NHL,other cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cellcarcinoma), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand Receptor1, lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma),VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumorassociated antigens recognized by antibodies have been reviewed (Gerber,et al, mAbs 1:3, 247-253 (2009); Novellino et al, Cancer ImmunolImmunother. 54(3), 187-207 (2005). Franke, et al, Cancer BiotherRadiopharm. 2000, 15, 459-76).

The cell-binding agents, more preferred antibodies, can be any agentsthat are able to against tumor cells, virus infected cells,microorganism infected cells, parasite infected cells, autoimmune cells,activated cells, myeloid cells, activated T-cells, B cells, ormelanocytes. More specifically the cell binding agents can be anyagent/molecule that is able to against any one of the following antigensor receptors: CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b,CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22,CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34,CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46,CD47, CD48, CD49b, CD49c, CD51, CD52, CD53, CD54, CD55, CD56, CD58,CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72,CD74, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD86, CD87, CD88,CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106, CD109,CD117, CD120, CD125, CD126, CD127, CD133, CD134, CD135, CD138, CD141,CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156, CD158,CD163, CD166, CD168, CD174, CD180, CD184, CDw186, CD194, CD195, CD200,CD200a, CD200b, CD209, CD221, CD227, CD235a, CD240, CD262, CD271, CD274,CD276 (B7-H3), CD303, CD304, CD309, CD326, 4-1BB, 5AC, 5T4 (Trophoblastglycoprotein, TPBG, 5T4, Wnt-Activated Inhibitory Factor 1 or WAIF 1),Adenocarcinoma antigen, AGS-5, AGS-22M6, Activin receptor-like kinase 1,AFP, AKAP-4, ALK, Alpha intergrin, Alpha v beta6, Amino-peptidase N,Amyloid beta, Androgen receptor, Angiopoietin 2, Angiopoietin 3, AnnexinA1, Anthrax toxin protective antigen, Anti-transferrin receptor, AOC3(VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF (B-cell activatingfactor), B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen,CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonicanhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonicanhydrase IX, Cardiac myosin, CCL11(C-C motif chemokine 11), CCR4 (C-Cchemokine receptor type 4, CD194), CCR5, CD3E (epsilon), CEA(Carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen),CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18),Clumping factor A, CRIPTO, FCSF1R (Colony stimulating factor 1 receptor,CD115), CSF2 (colony stimulating factor 2, Granulocyte-macrophagecolony-stimulating factor (GM-CSF)), CTLA4 (cytotoxicT-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4(CD184), C-X-C chemokine receptor type 4, cyclic ADP ribose hydrolase,Cyclin B1, CYP1B1, Cytomegalovirus, Cytomegalovirus glycoprotein B,Dabigatran, DLL4 (delta-like-ligand 4), DPP4 (Dipeptidyl-peptidase 4),DR5 (Death receptor 5), E. coli shiga toxin type-1, E. coli shiga toxintype-2, ED-B, EGFL7 (EGF-like domain-containing protein 7), EGFR,EGFRII, EGFRvIII, Endoglin (CD105), Endothelin B receptor, Endotoxin,EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2(Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS fusiongene), Escherichia coli, ETV6-AML, FAP (Fibroblast activation proteinalpha), FCGR1, alpha-Fetoprotein, Fibrin II, beta chain, Fibronectinextra domain-B, FOLR (folate receptor), Folate receptor alpha, Folatehydrolase, Fos-related antigen 1.F protein of respiratory syncytialvirus, Frizzled receptor, Fucosyl GM1, GD2 ganglioside, G-28 (a cellsurface antigen glyvolipid), GD3 idiotype, GloboH, Glypican 3,N-glycolylneuraminic acid, GM3, GMCSF receptor α-chain, Growthdifferentiation factor 8, GP100, GPNMB (Transmembrane glycoprotein NMB),GUCY2C (Guanylate cyclase 2C, guanylyl cyclase C(GC-C), intestinalGuanylate cyclase, Guanylate cyclase-C receptor, Heat-stable enterotoxinreceptor (hSTAR)), Heat shock proteins, Hemagglutinin, Hepatitis Bsurface antigen, Hepatitis B virus, HER1 (human epidermal growth factorreceptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (Hepatocytegrowth factor/scatter factor), HHGFR, HIV1, Histone complex, HLA-DR(human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, Human chorionicgonadotropin, HNGF, Human scatter factor receptor kinase, HPV E6/E7,Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype,IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ,Influeza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor(interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R (interleukin 6receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL22, IL-23,IL31RA, ILGF2 (Insulin-like growth factor 2), Integrins (α4, α_(IIb)β₃,αvβ3, α₄β₇, α5β1, α6β4, α7β7, αllβ3, α5β5, αvβ5), Interferongamma-induced protein, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Yantigen, LFA-1(Lymphocyte function-associated antigen 1, CD11a), LHRH,LINGO-1, Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2,MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF(Macrophage migration inhibitory factor, or glycosylation-inhibitingfactor (GIF)), MS4A1 (membrane-spanning 4-domains subfamily A member 1),MSLN (mesothelin), MUC1(Mucin 1, cell surface associated (MUC1) orpolymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125),MCP1(monocyte chemotactic protein 1), MelanA/MART1, ML-IAP, MPG, MS4A1(membrane-spanning 4-domains subfamily A), MYCN, Myelin-associatedglycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen),Nectin-4 (ASG-22ME), NGF, Neural apoptosis-regulated proteinase 1,NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1,NYESO-1, OX-40, OxLDL (Oxidized low-density lipoprotein), OY-TES1, P21,p53 nonmutant, P97, Page4, PAP, Paratope of anti-(N-glycolylneuraminicacid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, Programmed cell death protein 1,CD279), PDGF-Rα (Alpha-type platelet-derived growth factor receptor),PDGFR-Pβ, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase,Platelet-derived growth factor receptor beta, Phosphate-sodiumcotransporter, PMEL 17, Polysialic acid, Proteinase3 (PR1), Prostaticcarcinoma, PS (Phosphatidylserine), Prostatic carcinoma cells,Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD(Rh polypeptide 1 (RhPI), CD240), Rhesus factor, RANKL, RhoC, Rasmutant, RGS5, ROBO4, Respiratory syncytial virus, RON, Sarcomatranslocation breakpoints, SART3, Sclerostin, SLAMF7 (SLAM family member7), Selectin P, SDC1 (Syndecan 1), sLe(a), Somatomedin C, SIP(Sphingosine-1-phosphate), Somatostatin, Sperm protein 17, SSX2, STEAP1(six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn,TAG-72 (tumor associated glycoprotein 72), Survivin, T-cell receptor, Tcell transmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2,Tenascin C (TN-C), TGF-α, TGF-β (Transforming growth factor beta),TGF-β1, TGF-β2 (Transforming growth factor-beta 2), Tie (CD202b), Tie2,TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF1B (tumor necrosisfactor receptor superfamily member 10B), TNFRSF13B (tumor necrosisfactor receptor superfamily member 13B), TPBG (trophoblastglycoprotein), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligandReceptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calciumsignal transducer 2, tumor specific glycosylation of MUC1, TWEAKreceptor, TYRP1(glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD106),VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1,or cells expressing any insulin growth factor receptors, or anyepidermal growth factor receptors.

In another specific embodiment, the cell-binding ligand-drug conjugatesvia the bridge linkers of this invention are used for the targetedtreatment of cancers. The targeted cancers include, but are not limited,Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor(Adult, Brain Stem Glioma, Childhood, Cerebellar Astrocytoma, CerebralAstrocytoma, Ependymoma, Medulloblastoma, Supratentorial PrimitiveNeuroectodermal and Pineal Tumors, Visual Pathway and HypothalamicGlioma), Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma ofUnknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer,Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family ofTumors (PNET), Extracranial Germ Cell Tumor, Eye Cancer, IntraocularMelanoma, Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell Tumor,Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer,Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cellcancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid,Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and OralCavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell,Lymphoma, (AIDS-Related, Central Nervous System, Cutaneous T-Cell,Hodgkin's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma,Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer withOccult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms,Mycosis Fungoides, Myelodysplastic Syndrome, MyeloproliferativeDisorders, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer,Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, GermCell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine,Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer,Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, PituitaryCancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, RectalCancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter(Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, SkinCancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma,Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer,Testicular Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer,Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer,Vulvar Cancer, Wilms' Tumor.

In another specific embodiment, the cell-binding-drug conjugates via thebridge likers of this invention are used in accordance with thecompositions and methods for the treatment or prevention of anautoimmune disease. The autoimmune diseases include, but are notlimited, Achlorhydra Autoimmune Active Chronic Hepatitis, AcuteDisseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis,Addison's Disease, Agammaglobulinemia, Alopecia areata, AmyotrophicLateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis,Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopicallergy, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmunecardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis,Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome,Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmunepolyendocrine syndrome Types I, II, & III, Autoimmune progesteronedermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis,Balo disease/Balo concentric sclerosis, Bechets Syndrome, Berger'sdisease, Bickerstaffs encephalitis, Blau syndrome, Bullous Pemphigoid,Castleman's disease, Chagas disease, Chronic Fatigue Immune DysfunctionSyndrome, Chronic inflammatory demyelinating polyneuropathy, Chronicrecurrent multifocal ostomyelitis, Chronic lyme disease, Chronicobstructive pulmonary disease, Churg-Strauss syndrome, CicatricialPemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease,Complement component 2 deficiency, Cranial arteritis, CREST syndrome,Crohns Disease (a type of idiopathic inflammatory bowel diseases),Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease,Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetesmellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler'ssyndrome, Discoid lupus erythematosus, Eczema, Endometriosis,Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysisbullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia,Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia,Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinalpemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture'ssyndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, Haemolytic anaemia,Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa,Hughes syndrome (See Antiphospholipid syndrome), Hypogamma-globulinemia,Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonaryfibrosis, Idiopathic thrombocytopenic purpura (See Autoimmunethrombocytopenic purpura), IgA nephropathy (Also Berger's disease),Inclusion body myositis, Inflammatory demyelinating polyneuopathy,Interstitial cystitis, Irritable Bowel Syndrome, Juvenile idiopathicarthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease,Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichenplanus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease(Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupuserythematosus, Majeed syndrome, Ménière's disease, Microscopicpolyangiitis, Miller-Fisher syndrome, Mixed Connective Tissue Disease,Morphea, Mucha-Habermann disease, Muckle-Wells syndrome, MultipleMyeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy,Neuromyelitis optica (Devic's Disease), Neuromyotonia, Occularcicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis,Palindromic rheumatism, PANDAS (Pediatric Autoimmune NeuropsychiatricDisorders Associated with Streptococcus), Paraneoplastic cerebellardegeneration, Paroxysmal nocturnal hemoglobinuria, Parry Rombergsyndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus,Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis,POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica,Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis,Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis,Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome,Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis,Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome,Schnitzler syndrome, Scleritis, Scleroderma, Sjögren's syndrome,Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiffperson syndrome, Subacute bacterial endocarditis, Susac's syndrome,Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu'sarteritis, Temporal arteritis (giant cell arteritis), Tolosa-Huntsyndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathicinflammatory bowel diseases), Undifferentiated connective tissuedisease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo,Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome

In another specific embodiment, a binding molecule used for theconjugate via the bridge linkers of this invention for the treatment orprevention of an autoimmune disease can be, but are not limited to,anti-elastin antibody; Abys against epithelial cells antibody;Anti-Basement Membrane Collagen Type IV Protein antibody; Anti-NuclearAntibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG;anti-celiac antibody; Anti Phospholipid Antibody IgK, IgG; Anti SMAntibody; Anti Mitochondrial Antibody; Thyroid Antibody; MicrosomalAntibody, T-cells antibody; Thyroglobulin Antibody, Anti SCL-70;Anti-Jo; Anti-U.sub.1RNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti PeritalCells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anticentromere; AntiFibrillarin, and Anti GBM Antibody, Anti-gangliosideantibody; Anti-Desmogein 3 antibody; Anti-p62 antibody; Anti-sp100antibody; Anti-Mitochondrial(M2) antibody; Rheumatoid factor antibody;Anti-MCV antibody; Anti-topoisomerase antibody; Antineutrophilcytoplasmic(cANCA) antibody.

In certain preferred embodiments, the binding molecule for the conjugatein the present invention, can bind to both a receptor and a receptorcomplex expressed on an activated lymphocyte which is associated with anautoimmune disease. The receptor or receptor complex can comprise animmunoglobulin gene superfamily member (e.g. CD2, CD3, CD4, CD8, CD19,CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90,CD125, CD147, CD152/CTLA-4, PD-1, or ICOS), a TNF receptor superfamilymember (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1,TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2,TRAIL-R3, TRAIL-R4, and APO-3), an integrin, a cytokine receptor, achemokine receptor, a major histocompatibility protein, a lectin(C-type, S-type, or I-type), or a complement control protein.

In another specific embodiment, useful cell binding ligands that areimmunospecific for a viral or a microbial antigen are humanized or humanmonoclonal antibodies. As used herein, the term “viral antigen”includes, but is not limited to, any viral peptide, polypeptide protein(e.g. HIV gp120, HIV nef, RSV F glycoprotein, influenza virusneuramimidase, influenza virus hemagglutinin, HTLV tax, herpes simplexvirus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surfaceantigen) that is capable of eliciting an immune response. As usedherein, the term “microbial antigen” includes, but is not limited to,any microbial peptide, polypeptide, protein, saccharide, polysaccharide,or lipid molecule (e.g., a bacteria, fungi, pathogenic protozoa, oryeast polypeptides including, e.g., LPS and capsular polysaccharide 5/8)that is capable of eliciting an immune response. Examples of antibodiesavailable 1 for the viral or microbial infection include, but are notlimited to, Palivizumab which is a humanized anti-respiratory syncytialvirus monoclonal antibody for the treatment of RSV infection; PRO542which is a CD4 fusion antibody for the treatment of HIV infection;Ostavir which is a human antibody for the treatment of hepatitis Bvirus; PROTVIR which is a humanized IgG.sub.1 antibody for the treatmentof cytomegalovirus; and anti-LPS antibodies.

The cell binding molecules-drug conjugates via the bridge linkers ofthis invention can be used in the treatment of infectious diseases.These infectious diseases include, but are not limited to, Acinetobacterinfections, Actinomycosis, African sleeping sickness (Africantrypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis,Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentinehemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection,Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterialvaginosis, Bacteroides infection, Balantidiasis, Baylisascarisinfection, BK virus infection, Black piedra, Blastocystis hominisinfection, Blastomycosis, Bolivian hemorrhagic fever, Borreliainfection, Botulism (and Infant botulism), Brazilian hemorrhagic fever,Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection(Norovirus and Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis;Thrush), Cat-scratch disease, Cellulitis, Chagas Disease (Americantrypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydophilapneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis,Clostridium difficile infection, Coccidioidomycosis, Colorado tickfever, Common cold (Acute viral rhinopharyngitis; Acute coryza),Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever,Cryptococ-cosis, Cryptosporidiosis, Cutaneous larva migrans,Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever,Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebolahemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworminfection), Enterococcus infection, Enterovirus infection, Epidemictyphus, Erythema infectiosum (Fifth disease), Exanthem subitum,Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Foodpoisoning by Clostridium perfringens, Free-living amebic infection,Fusobacterium infection, Gas gangrene (Clostridial myonecrosis),Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis,Glanders, Gnathosto-miasis, Gonorrhea, Granuloma inguinale(Donovanosis), Group A streptococcal infection, Group B streptococcalinfection, Haemophilus influenzae infection, Hand, foot and mouthdisease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pyloriinfection, Hemolytic-uremic syndrome, Hemorrhagic fever with renalsyndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, HepatitisE, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirusinfection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis,Human metapneumovirus infection, Human monocytic ehrlichiosis, Humanpapillomavirus infection, Human parainfluenza virus infection,Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono),Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingaeinfection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease),Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis,Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis(Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburghemorrhagic fever, Measles, Melioidosis (Whitmore's disease),Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis,Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasmapneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmianeonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD),Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis(South American blastomycosis), Paragonimiasis, Pasteurellosis,Pediculosis capitis (Head lice), Pediculosis corporis (Body lice),Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease,Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystispneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primaryamoebic meningoencephalitis, Progressive multifocal leukoencephalopathy,Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytialvirus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsialinfection, Rickettsial-pox, Rift Valley fever, Rocky mountain spottedfever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe AcuteRespiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis(Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola),Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection,Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae(Barber's itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis(Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringwormof the Hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium(Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis(Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans),Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworminfection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection,Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viralpneumonia, West Nile Fever, White piedra (Tinea blanca), Yersiniapseudotuber-culosis infection, Yersiniosis, Yellow fever, Zygomycosis.

The cell binding molecule, which is more preferred to be an antibodydescribed in this patent that are against pathogenic strains include,but are not limit, Acinetobacter baumannii, Actinomyces israelii,Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosomabrucei, HIV (Human immunodeficiency virus), Entamoeba histolytica,Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Juninvirus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family,Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus,Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae,Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borreliagenus, Clostridium botulinum, Sabia, Brucella genus, usuallyBurkholderia cepacia and other Burkholderia species, Mycobacteriumulcerans, Caliciviridae family, Campylobacter genus, usually Candidaalbicans and other Candida species, Bartonella henselae, Group AStreptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilusducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis,Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi,Clonorchis sinensis, Clostridium difficile, Coccidioides immitis andCoccidioides posadasii, Colorado tick fever virus, rhinoviruses,coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever virus,Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma braziliense;multiple parasites, Cyclospora cayetanensis, Taenia solium,Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 andDEN-4)—Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae,Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcusgenus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus,Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Humanherpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciolahepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily,Clostridium perfringens, Fusobacterium genus, Clostridium perfringens;other Clostridium species, Geotrichum candidum, GSS prion, Giardiaintestinalis, Burkholderia mallei, Gnathostoma spinigerum andGnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis,Streptococcus pyogenes, Streptococcus agalactiae, Haemophilusinfluenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71,Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7,Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis CVirus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1,Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenaleand Necator americanus, Hemophilus influenzae, Human bocavirus,Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus,Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenzaviruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus,Orthomyxoviridae family, Isospora belli, Kingella kingae, Klebsiellapneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion,Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmaniagenus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospiragenus, Listeria monocytogenes, Borrelia burgdorferi and other Borreliaspecies, Wuchereria bancrofti and Brugia malayi, Lymphocyticchoriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measlesvirus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous speciesof bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterousfly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion,Nocardia asteroides and other Nocardia species, Onchocerca volvulus,Paracoccidioides brasiliensis, Paragonimus westermani and otherParagonimus species, Pasteurella genus, Pediculus humanus capitis,Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis,Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii,Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophilapsittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformisand Spirillum minus, Respiratory syncytial virus, Rhinosporidiumseeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valleyfever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonellagenus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigellagenus, Varicella zoster virus, Variola major or Variola minor,Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus,Staphylococcus aureus, Streptococcus pyogenes, Strongyloidesstercoralis, Treponema pallidum, Taenia genus, Clostridium tetani,Trichophyton genus, Trichophyton tonsurans, Trichophyton genus,Epidermophyton floccosum, Trichophyton rubrum, and Trichophytonmentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophytongenus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasmagondii, Trichinella spiralis, Trichomonas vaginalis, Trichuristrichiura, Mycobacterium tuberculosis, Francisella tularensis,Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibriocolerae, Guanarito virus, West Nile virus, Trichosporon beigelii,Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fevervirus, Mucorales order (Mucormycosis) and Entomophthorales order(Entomophthora-mycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio)fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis,Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonellatyphimurium, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Pneumocystis carinii, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus,Candida albicans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosomagambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmaniatropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodiumvivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths(Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium,and hookworms).

Other antibodies as cell binding ligands used in this invention fortreatment of viral disease include, but are not limited to, antibodiesagainst antigens of pathogenic viruses, including as examples and not bylimitation: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae,Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae,influenza viruses, parainfluenza viruses, mumps, measles, respiratorysyncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae,Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae,Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervicalcancer, Anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi'ssarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt'slymphoma, Primary central nervous system lymphoma), MCPyV (Merkel cellcancer), SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I(Adult T-cell leukemia/lymphoma)], Immune disorders caused virus: [suchas Human Immunodeficiency Virus (AIDS)]; Central nervous system virus:[such as, JCV (Progressive multifocal leukoencephalopathy), MeV(Subacute sclerosing panencephalitis), LCV (Lymphocyticchoriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable)(Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviralmeningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis,Postpolio syndrome), HTLV-I (Tropical spastic paraparesis)];Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis));Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)];Respiratory system/acute viral nasopharyngitis/viral pneumonia:[Epstein-Barr virus (EBV infection/Infectious mononucleosis),Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome)Orthomyxoviridae: Influenzavirus A/B/C (Influenza/Avian influenza),Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Humanrespiratory syncytial virus), hMPV]; Digestive system virus [MuV(Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus(Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus;HBV (Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis Cvirus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (HepatitisG virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)].

According to a further object, the present invention also concernspharmaceutical compositions comprising the conjugate via the bridgelinkers of the invention together with a pharmaceutically acceptablecarrier, diluent, or excipient for treatment of cancers, infections orautoimmune disorders. The method for treatment of cancers, infectionsand autoimmune disorders can be practiced in vitro, in vivo, or ex vivo.Examples of in vitro uses include treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.Examples of ex vivo uses include treatments of hematopoietic stem cells(HSC) prior to the performance of the transplantation (HSCT) into thesame patient in order to kill diseased or malignant cells. For instance,clinical ex vivo treatment to remove tumour cells or lymphoid cells frombone marrow prior to autologous transplantation in cancer treatment orin treatment of autoimmune disease, or to remove T cells and otherlymphoid cells from allogeneic bone marrow or tissue prior to transplantin order to prevent graft-versus-host disease, can be carried out asfollows. Bone marrow is harvested from the patient or other individualand then incubated in medium containing serum to which is added theconjugate of the invention, concentrations range from about 1 pM to 0.1mM, for about 30 minutes to about 48 hours at about 37° C. The exactconditions of concentration and time of incubation (=dose) are readilydetermined by the skilled clinicians. After incubation, the bone marrowcells are washed with medium containing serum and returned to thepatient by i.v. infusion according to known methods. In circumstanceswhere the patient receives other treatment such as a course of ablativechemotherapy or total-body irradiation between the time of harvest ofthe marrow and reinfusion of the treated cells, the treated marrow cellsare stored frozen in liquid nitrogen using standard medical equipment.

For clinical in vivo use, the conjugate via the linkers of the inventionwill be supplied as solutions or as a lyophilized solid that can beredissolved in sterile water for injection. Examples of suitableprotocols of conjugate administration are as follows. Conjugates aregiven weekly for 8˜20 weeks as an i.v. bolus. Bolus doses are given in50 to 500 ml of normal saline to which human serum albumin (e.g. 0.5 to1 mL of a concentrated solution of human serum albumin, 100 mg/mL) canbe added. Dosages will be about 50 μg to 20 mg/kg of body weight perweek, i.v. (range of 10 μg to 200 mg/kg per injection). 4˜20 weeks aftertreatment, the patient may receive a second course of treatment.Specific clinical protocols with regard to route of administration,excipients, diluents, dosages, times, etc., can be determined by theskilled clinicians.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of killing selected cell populations includemalignancy of any types of cancer, autoimmune diseases, graftrejections, and infections (viral, bacterial or parasite).

The amount of a conjugate which is required to achieve the desiredbiological effect, will vary depending upon a number of factors,including the chemical characteristics, the potency, and thebioavailability of the conjugates, the type of disease, the species towhich the patient belongs, the diseased state of the patient, the routeof administration, all factors which dictate the required dose amounts,delivery and regimen to be administered.

In general terms, the conjugates via the linkers of this invention maybe provided in an aqueous physiological buffer solution containing 0.1to 10% w/v conjugates for parenteral administration. Typical dose rangesare from 1 μg/kg to 0.1 g/kg of body weight per day; a preferred doserange is from 0.01 mg/kg to 20 mg/kg of body weight per day, or perweek, or an equivalent dose in a human child. The preferred dosage ofdrug to be administered is likely to depend on such variables as thetype and extent of progression of the disease or disorder, the overallhealth status of the particular patient, the relative biologicalefficacy of the compound selected, the formulation of the compound, theroute of administration (intravenous, intramuscular, or other), thepharmacokinetic properties of the conjugates by the chosen deliveryroute, and the speed (bolus or continuous infusion) and schedule ofadministrations (number of repetitions in a given period of time).

The conjugates via the linkers of the present invention are also capableof being administered in unit dose forms, wherein the term “unit dose”means a single dose which is capable of being administered to a patient,and which can be readily handled and packaged, remaining as a physicallyand chemically stable unit dose comprising either the active conjugateitself, or as a pharmaceutically acceptable composition, as describedhereinafter. As such, typical total daily/weekly/biweekly/monthly doseranges are from 0.01 to 100 mg/kg of body weight. By way of generalguidance, unit doses for humans range from 1 mg to 3000 mg per day, orper week, per two week or per month. Preferably the unit dose range isfrom 1 to 500 mg administered one to four times a week, and even morepreferably from 1 mg to 100 mg, once a week. Conjugates provided hereincan be formulated into pharmaceutical compositions by admixture with oneor more pharmaceutically acceptable excipients. Such unit dosecompositions may be prepared for use by oral administration,particularly in the form of tablets, simple capsules or soft gelcapsules; or intranasal, particularly in the form of powders, nasaldrops, or aerosols; or dermally, for example, topically in ointments,creams, lotions, gels or sprays, or via trans-dermal patches.

Drugs/Cytotoxic Agents

Drugs that can be conjugated to a cell-binding molecule in the presentinvention are small molecule drugs including cytotoxic agents, which canbe linked to or after they are modified for linkage to the cell-bindingagent. A “small molecule drug” is broadly used herein to refer to anorganic, inorganic, or organometallic compound that may have a molecularweight of for example 100 to 1800, more suitably from 120 to 1400. Smallmolecule drugs are well characterized in the art, such as inWO05058367A2, and in U.S. Pat. No. 4,956,303, among others and areincorporated in their entirety by reference. The drugs include knowndrugs and those that may become known drugs.

Drugs that are known include, but not limited to,

1). Chemotherapeutic agents: a). Alkylating agents: such as Nitrogenmustards: chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, mannomustine, mitobronitol, melphalan, mitolactol,pipobroman, novembichin, phenesterine, prednimustine, thiotepa,trofosfamide, uracil mustard; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); Duocarmycin (includingthe synthetic analogues, KW-2189 and CBI-TMI); Benzodiazepine dimers(e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin,indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidinobenzodiazepines); Nitrosoureas: (carmustine, lomustine,chlorozotocin, fotemustine, nimustine, ranimustine); Alkylsulphonates:(busulfan, treosulfan, improsulfan and piposulfan); Triazenes:(dacarbazine); Platinum containing compounds: (carboplatin, cisplatin,oxaliplatin); aziridines, such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemel-amine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomel-amine]; b). PlantAlkaloids: such as Vinca alkaloids: (vincristine, vinblastine,vindesine, vinorelbine, navelbin); Taxoids: (paclitaxel, docetaxol) andtheir analogs, Maytansinoids (DM1, DM2, DM3, DM4, maytansine andansamitocins) and their analogs, cryptophycins (particularlycryptophycin 1 and cryptophycin 8); epothilones, eleutherobin,discodermo-lide, bryostatins, dolostatins, auristatins, tubulysins,cephalostatins; pancratistatin; a sarcodictyin; spongistatin; c). DNATopoisomerase Inhibitors: such as [Epipodophyllins:(9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide,etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoicacids (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS2000)); mitomycins: (mitomycin C)]; d). Anti-metabolites: such as{[Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate, denopterin,pteropterin, aminopterin (4-aminopteroic acid) or the other folic acidanalogues); IMP dehydrogenase Inhibitors: (mycophenolic acid,tiazofurin, ribavirin, EICAR); Ribonucleotide reductase Inhibitors:(hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs:(ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil,floxuridine, ratitrexed (Tomudex)); Cytosine analogs: (cytarabine,cytosine arabinoside, fludarabine); Purine analogs: (azathioprine,fludarabine, mercaptopurine, thiamiprine, thioguanine)]; folic acidreplenisher, such as frolinic acid}; e). Hormonal therapies: such as{Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene,tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate);Anti-androgens: (bicalutamide, flutamide, calusterone, dromostanolonepropionate, epitiostanol, goserelin, leuprolide, mepitiostane,nilutamide, testolactone, trilostane and other androgens inhibitors)];Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060,cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin,phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A);Cytokines: (Interferon-alpha, Interferon-gamma, tumor necrosis factor(TNFs), human proteins containing a TNF domain)]}; f). Kinaseinhibitors, such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib,pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib,lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2),mubritinib, ponatinib (AP24534), bafetinib (INNO406), bosutinib(SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387,axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab,Ranibizumab, Panitumumab, ispinesib; g). antibiotics, such as theenediyne antibiotics (e.g. calicheamicins, especially calicheamicin γ1,δ1, α1 and β1, see, e.g., J. Med. Chem., 39 (11), 2103-2117 (1996),Angew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicin, includingdynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027,maduropeptin, as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin; chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; f). Others: such as Polyketides (acetogenins),especially bullatacin and bullatacinone; gemcitabine, epoxomicins (e. g.carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide,tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7,Xegeva, Provenge, Yervoy, Isoprenylation inhibitors (such asLovastatin), Dopaminergic neurotoxins (such as1-methyl-4-phenylpyridinium ion), Cell cycle inhibitors (such asstaurosporine), Actinomycins (such as Actinomycin D, dactinomycin),Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin),Anthracyclines (such as daunorubicin, doxorubicin (adriamycin),idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDRinhibitors (such as verapamil), Ca²⁺ ATPase inhibitors (such asthapsigargin), Histone deacetylase inhibitors (Vorinostat, Romidepsin,Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat,PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101,sulforaphane, Trichostatin A); Thapsigargin, Celecoxib, glitazones,epigallocatechin gallate, Disulfiram, Salinosporamide A.; Anti-adrenals,such as aminoglutethimide, mitotane, trilostane; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside,bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; eflornithine (DFMO), elfomithine; elliptinium acetate,etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate,lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verrucarin A, roridin A and anguidine); urethane, siRNA, antisensedrugs, and a nucleolytic enzyme.

2). An anti-autoimmune disease agent includes, but is not limited to,cyclosporine, cyclosporine A, aminocaproic acid, azathioprine,bromocriptine, chlorambucil, chloroquine, cyclophosphamide,corticosteroids (e.g. amcinonide, betamethasone, budesonide,hydrocortisone, flunisolide, fluticasone propionate, fluocortolonedanazol, dexamethasone, Triamcinolone acetonide, beclometasonedipropionate), DHEA, enanercept, hydroxychloroquine, infliximab,meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus,tacrolimus.

3). An anti-infectious disease agent includes, but is not limited to,a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin,sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin,bekanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin,ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin,verdamicin; b). Amphenicols: azidamfenicol, chloramphenicol,florfenicol, thiamphenicol; c). Ansamycins: geldanamycin, herbimycin;d). Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin,meropenem, panipenem; e). Cephems: carbacephem (loracarbef),cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine,cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole,cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin,cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin,cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir,cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin,cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome,cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime,ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin,cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f).Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin),teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. g.tigecycline; g). β-Lactamase inhibitors: penam (sulbactam, tazobactam),clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j).Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA);k). Macrolides: azithromycin, cethromycin, clarithromycin,dirithromycin, erythromycin, flurithromycin, josamycin, ketolide(telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin,rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin,roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506),troleandomycin, telithromycin; l). Monobactams: aztreonam, tigemonam;m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin(pivampicillin, hetacillin, bacampicillin, metampicillin,talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathinebenzylpenicillin, benzathine phenoxymethyl-penicillin, clometocillin,procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin,dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam),mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin,pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin,sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin,colistin, polymyxin B; p). Quinolones: alatrofloxacin, balofloxacin,ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin,enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin,grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin,marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin,ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin,sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q).Streptogramins: pristinamycin, quinupristin/dalfopristin); r).Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole,sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole); s). Steroidantibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline,chlortetracycline, clomocycline, demeclocycline, lymecycline,meclocycline, metacycline, minocycline, oxytetracycline,penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g.tigecycline); u). Other types of antibiotics: annonacin, arsphenamine,bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine),dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol,etoposide, faropenem, fusidic acid, furazolidone, isoniazid,laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesisinhibitors (e. g. fosfomycin), nitrofurantoin, paclitaxel,platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin(rifampin), tazobactam tinidazole, uvaricin;

4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc,vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integraseinhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturationinhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors:oseltamivir, zanamivir, peramivir; e). Nucleosides &nucleotides:abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine,cidofovir, clevudine, dexelvucitabine, didanosine (ddI), elvucitabine,emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU),3′-fluoro-substituted 2′, 3′-dideoxynucleoside analogues (e.g.3′-fluoro-2′,3′-dideoxythymidine (FLT) and3′-fluoro-2′,3′-dideoxyguanosine (FLG), fomivirsen, ganciclovir,idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. β-1-thymidine andβ-1-2′-deoxycytidine), penciclovir, racivir, ribavirin, stampidine,stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir,trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine(AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine,diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol,emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod,interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205,peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod(R-848), tromantadine; g). Protease inhibitors: amprenavir, atazanavir,boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir,pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h).Other types of anti-virus drugs: abzyme, arbidol, calanolide a,ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate(EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea,KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin,seliciclib.

5). The drugs used for conjugates via a bridge linker of the presentinvention also include radioisotopes. Examples of radioisotopes(radionuclides) are ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴CU, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, or ²¹³Bi.Radioisotope labeled antibodies are useful in receptor targeted imagingexperiments or can be for targeted treatment such as with theantibody-drug conjugates of the invention (Wu et al (2005) NatureBiotechnology 23(9): 1137-1146). The cell binding molecules, e.g. anantibody can be labeled with ligand reagents through the bridge linkersof the present patent that bind, chelate or otherwise complex aradioisotope metal, using the techniques described in Current Protocolsin Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience,New York, N.Y., Pubs. (1991). Chelating ligands which may complex ametal ion include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics,Dallas, Tex. USA).

6). The pharmaceutically acceptable salts, acids or derivatives of anyof the above drugs.

In another embodiment, the drug in the Formula (II) and (IV) can achromophore molecule, for which the conjugate can be used for detection,monitoring, or study the interaction of the cell binding molecule with atarget cell. Chromophore molecules are a compound that have the abilityto absorb a kind of light, such as UV light, florescent light, IR light,near IR light, visual light; A chromatophore molecule includes a classor subclass of xanthophores, erythrophores, iridophores, leucophores,melanophores, and cyanophores; a class or subclass of fluorophoremolecules which are fluorescent chemical compounds reemitting light uponlight; a class or subclass of visual phototransduction molecules; aclass or subclass of photophore molecules; a class or subclass ofluminescence molecules; and a class or subclass of luciferin compounds.

The chromophore molecule can be selected from, but not limited,Non-protein organic fluorophores, such as: Xanthene derivatives(fluorescein, rhodamine, Oregon green, eosin, and Texas red); Cyaninederivatives: (cyanine, indocarbocyanine, oxacarbocyanine,thiacarbocyanine, and merocyanine); Squaraine derivatives andring-substituted squaraines, including Seta, SeTau, and Square dyes;Naphthalene derivatives (dansyl and prodan derivatives); Coumarinderivatives; Oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazoleand benzoxadiazole); Anthracene derivatives (anthraquinones, includingDRAQ5, DRAQ7 and CyTRAK Orange); Pyrene derivatives (cascade blue, etc);Oxazine derivatives (Nile red, Nile blue, cresyl violet, oxazine 170etc). Acridine derivatives (proflavin, acridine orange, acridine yellowetc). Arylmethine derivatives (auramine, crystal violet, malachitegreen). Tetrapyrrole derivatives (porphin, phthalocyanine, bilirubin).

Or a chromophore molecule can be selected from any analogs andderivatives of the following fluorophore compounds: CF dye (Biotium),DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), Alexa Fluor(Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy(Sigma Aldrich), FluoProbes (Interchim), Abberior Dyes (Abberior), DYand MegaStokes Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor(AnaSpec), Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar andCal Fluor dyes (Biosearch Technologies), SureLight Dyes (APC, RPEPerCP,Phycobilisomes)(Columbia Biosciences), APC, APCXL, RPE, BPE(Phyco-Biotech);

Examples of the widely used fluorophore compounds which are reactive orconjugatable with the linkers of the invention are: Allophycocyanin(APC), Aminocou-marin, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2,Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin,Lissamine Rhodamine B, Lucifer yellow, Methoxycoumarin, NBD, PacificBlue, Pacific Orange, PE-Cy5 conjugates, PE-Cy7 conjugates, PerCP,R-Phycoerythrin(PE), Red 613, Seta-555-Azide, Seta-555-DBCO,Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-780-NHS, Seta-APC-780,Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405-Maleimide,SeTau-405-NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed,X-Rhodamine.

The fluorophore compounds that can be linked to the linkers of theinvention for study of nucleic acids or proteins are selected from thefollowing compounds or their derivatives: 7-AAD (7-aminoactinomycin D,CG-selective), Acridine Orange, Chromomycin A3, CyTRAK Orange(Biostatus, red excitation dark), DAPI, DRAQ5, DRAQ7, Ethidium Bromide,Hoechst33258, Hoechst33342, LDS 751, Mithramycin, Propidiumlodide (PI),SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO: CyanineMonomer, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. Thefluorophore compounds that can be linked to the linkers of the inventionfor study cells are selected from the following compounds or theirderivatives: DCFH (2′7′Dichorodihydro-fluorescein, oxidized form), DHR(Dihydrorhodamine 123, oxidized form, light catalyzes oxidation), Fluo-3(AM ester. pH >6), Fluo-4 (AM ester. pH 7.2), Indo-1 (AM ester, low/highcalcium (Ca2+)), SNARF(pH 6/9). The preferred fluorophore compounds thatcan be linked to the linkers of the invention for studyproteins/antibodies are selected from the following compounds or theirderivatives: Allophycocyanin(APC), AmCyan1 (tetramer, Clontech), AsRed2(tetramer, Clontech), Azami Green (monomer, MBL), Azurite,B-phycoerythrin(BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsRed2(“RFP”, Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech),Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65Amutation), GFP (S65C mutation), GFP (S65L mutation), GFP (S65Tmutation), GFP (Y66F mutation), GFP (Y66H mutation), GFP (Y66Wmutation), GFPuv, HcRed1, J-Red, Katusha, Kusabira Orange (monomer,MBL), mCFP, mCherry, mCitrine, Midoriishi Cyan (dimer, MBL), mKate(TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange,mPlum, mRaspberry, mRFP1 (monomer, Tsien lab), mStrawberry, mTFP1,mTurquoise2, P3 (phycobilisome complex), Peridinin Chlorophyll (PerCP),R-phycoerythrin(RPE), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP(dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen),tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFP635(dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen),TurboYFP (dimer, Evrogen), Venus, Wild Type GFP, YPet, ZsGreen1(tetramer, Clontech), ZsYellow1 (tetramer, Clontech).

In yet another embodiment, the preferred cytotoxic agents thatconjugated to a cell-binding molecule via a bridge linker of this patentare tubulysins, maytansinoids, taxanoids (taxanes), CC-1065 analogs,daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g.,dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin,indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidino-benzodiazepines), calicheamicins and the enediyneantibiotics, actinomycin, azaserines, bleomycins, epirubicin, tamoxifen,idarubicin, dolastatins, auristatins (e.g. monomethyl auristatin E,MMAE, MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB(AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines,hemiasterlins, nazumamides, microginins, radiosumins, alterobactins,microsclerodermins, theonellamides, esperamicins, PNU-159682, and theiranalogues and derivatives above thereof.

Tubulysins that are preferred for conjugation in the present inventionare well known in the art and can be isolated from natural sourcesaccording to known methods or prepared synthetically according to knownmethods (e. g. Balasubramanian, R.; et al. J. Med. Chem., 2009, 52,238-240. Wipf, P.; et al. Org. Lett., 2004, 6, 4057-4060. Pando, O.; etal. J. Am. Chem. Soc., 2011, 133, 7692-7695. Reddy, J. A.; et al. Mol.Pharmaceutics, 2009, 6, 1518-1525. Raghavan, B.; et al. J. Med. Chem.,2008, 51, 1530-1533. Patterson, A. W.; et al. J. Org. Chem., 2008, 73,4362-4369. Pando, O.; et al. Org. Lett., 2009, 11 (24), pp 5567-5569.Wipf, P.; et al. Org. Lett., 2007, 9 (8), 1605-1607. Friestad, G. K.;Org. Lett., 2004, 6, pp 3249-3252. Hillary M. Peltier, H. M.; et al. J.Am. Chem. Soc., 2006, 128, 16018-16019. Chandrasekhar, S.; et al. J.Org. Chem., 2009, 74, 9531-9534. Liu, Y.; et al. Mol. Pharmaceutics,2012, 9, 168-175. Friestad, G. K.; et al. Org. Lett., 2009, 11,1095-1098. Kubicek, K.; et al., Angew Chem Int Ed Engl, 2010. 49: p.4809-12. Chai, Y.; et al., Chem Biol, 2010, 17: 296-309. Ullrich, A.; etal., Angew Chem Int Ed Engl, 2009, 48, 4422-5. Sani, M.; et al. AngewChem Int Ed Engl, 2007, 46, 3526-9. Domling, A.; et al., Angew Chem IntEd Engl, 2006. 45, 7235-9. Patent applications: Zanda, M.; et al, Can.Pat. Appl. CA 2710693 (2011). Chai, Y.; et al. Eur. Pat. Appl. 2174947(2010), PCT WO 2010034724. Leamon, C.; et al, PCT WO 2010033733, WO2009002993. Ellman, J.; et al, PCT WO 2009134279; PCT WO 2009012958, USappl. 20110263650, 20110021568, Matschiner, G.; et al, PCT WO2009095447.Vlahov, I.; et al, PCT WO 2009055562, WO 2008112873. Low, P.;et al, PCT WO 2009026177. Richter, W., PCT WO 2008138561. Kjems, J.; etal, PCT WO 2008125116. Davis, M.; et al, PCT WO 2008076333. Diener, J.;et al, U.S. Pat. Appl. 20070041901, WO 2006096754. Matschiner, G.; etal, PCT WO 2006056464. Vaghefi, F.; et al, 5 PCT WO 2006033913.Doemling, A., Ger. Offen. DE 102004030227; PCT WO 2004005327; WO2004005326; WO2004005269. Stanton, M.; et al, U.S. Pat. Appl. Publ.20040249130. Hoefle, G.; et al, Ger. Offen. DE 10254439; DE 10241152; DE10008089. Leung, D.; et al, WO 2002077036. Reichenbach, H.; et al, Ger.Offen. DE 19638870; Wolfgang, R.; US 20120129779, Chen, H., US appl.20110027274. The preferred structure of tubulysins for conjugation ofcell binding molecules are described in the patent application ofPCT/IB2012/053554.

Examples of the structures of the conjugates of the antibody-tubulysinanalogs via the bridge linker are T01, T02, T03, T04, T05, T06 and T07as following:

Wherein mAb is an antibody; Z₃ and Z′₃ are independently HOP(O)(OM₁)(OM₂), OCH₂OP(O)(OM₁)(OM₂), OSO₃M1, R₁, or O-glycoside(glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside,etc), NH-glycoside, S-glycoside or CH₂-glycoside; M₁ and M₂ areindependently H, Na, K, Ca, Mg, NH₄, NR₁R2R₃; n is 1˜20; X₁, X₂, R₁, R₂and R₃ are the same defined in Formula (I).

Calicheamicins and their related enediyne antibiotics that are preferredfor cell-binding molecule-drug conjugates of this patent are describedin: Nicolaou, K. C. et al, Science 1992, 256, 1172-1178; Proc. Natl.Acad. Sci USA. 1993, 90, 5881-5888), U.S. Pat. Nos. 4,970,198;5,053,394; 5,108,912; 5,264,586; 5,384,412; 5,606,040; 5,712,374;5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 5,877,296;6,015,562; 6,124,310; 8,153,768. An Example of the structure of theconjugate of the antibody-Calicheamicin analog via the bridge linker isC01 as the following:

Wherein mAb is an antibody; n is 1˜20; X₁, X₂, R₁, R₂ and R₃ are thesame defined in Formula (I).

Maytansinoids that are preferred to be used in the present inventionincluding maytansinol and its analogues are described in U.S. Pat. Nos.4,256,746, 4,361,650, 4,307,016, 4,294,757, 4,294,757, 4,371,533,4,424,219, 4,331,598, 4,450,254, 4,364,866, 4,313,946, 4,315,9294,362,663, 4,322,348, 4,371,533, 4,424,219, 5,208,020, 5,416,064,5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821, 7,276,497,7,301,019, 7,303,749, 7,368,565, 7,411,063, 7,851,432, and 8,163,888. Anexample of the structure of the conjugate of the antibody-Maytansinoidsvia the bridge linker is as the following M01:

Wherein mAb is an antibody; n is 1˜20; X₁, X₂, R₁, R₂ and R₃ are thesame defined in Formula (I).

Taxanes, which includes Paclitaxel (Taxol), a cytotoxic natural product,and docetaxel (Taxotere), a semi-synthetic derivative, and their analogswhich are preferred for conjugation via the bridge linkers of thepresent patent are exampled in: K C. Nicolaou et al., J. Am. Chem. Soc.117, 2409-2420, (1995); Ojima et al, J. Med. Chem. 39:3889-3896 (1996);40:267-278 (1997); 45, 5620-5623 (2002); Ojima et al., Proc. Natl. Acad.Sci., 96:4256-4261 (1999; Kim et al., Bull. Korean Chem. Soc., 20,1389-1390 (1999); Miller, et al. J. Med. Chem., 47, 4802-4805(2004);U.S. Pat. Nos. 5,475,011 5,728,849, 5,811,452; 6,340,701; 6,372,738;6,391,913, 6,436,931; 6,589,979; 6,596,757; 6,706,708; 7,008,942;7,186,851; 7,217,819; 7,276,499; 7,598,290; and 7,667,054.

Examples of the structures of the conjugate of the antibody-taxanes viathe bridge linker are as the following Tx01, Tx02 and Tx03.

Wherein mAb is an antibody; n is 1˜20; X₁, X₂, R₁ and R₂ are the samedefined in Formula (I).

CC-1065 analogues and doucarmycin analogs are also preferred to be usedfor a conjugate with the bridge linkers of the present patent. Theexamples of the CC-1065 analogues and doucarmycin analogs as well astheir synthesis are described in: e.g. Warpehoski, et al, J. Med. Chem.31:590-603 (1988), D. Boger et al., J. Org. Chem; 66; 6654-6661, 2001;U.S. Pat. Nos. 4,169,888, 4,391,904, 4,671,958, 4,816,567, 4,912,227,4,923,990, 4,952,394, 4,975,278, 4,978,757, 4,994,578, 5,037,993,5,070,092, 5,084,468, 5,101,038, 5,117,006, 5,137,877, 5,138,059,5,147,786, 5,187,186, 5,223,409, 5,225,539, 5,288,514, 5,324,483,5,332,740, 5,332,837, 5,334,528, 5,403,484, 5,427,908, 5,475,092,5,495,009, 5,530,101, 5,545,806, 5,547,667, 5,569,825, 5,571,698,5,573,922, 5,580,717, 5,585,089, 5,585,499, 5,587,161, 5,595,499,5,606,017, 5,622,929, 5,625,126, 5,629,430, 5,633,425, 5,641,780,5,660,829, 5,661,016, 5,686,237, 5,693,762, 5,703,080, 5,712,374,5,714,586, 5,739,116, 5,739,350, 5,770,429, 5,773,001, 5,773,435,5,786,377 5,786,486, 5,789,650, 5,814,318, 5,846,545, 5,874,299,5,877,296, 5,877,397, 5,885,793, 5,939,598, 5,962,216, 5,969,108,5,985,908, 6,060,608, 6,066,742, 6,075,181, 6,103,236, 6,114,598,6,130,237, 6,132,722, 6,143,901, 6,150,584, 6,162,963, 6,172,197,6,180,370, 6,194,612, 6,214,345, 6,262,271, 6,281,354, 6,310,209,6,329,497, 6,342,480, 6,486,326, 6,512,101, 6,521,404, 6,534,660,6,544,731, 6,548,530, 6,555,313, 6,555,693, 6,566,336, 6,586,618,6,593,081, 6,630,579, 6,756,397, 6,759,509, 6,762,179, 6,884,869,6,897,034, 6,946,455, 7,049,316, 7,087,600, 7,091,186, 7,115,573,7,129,261, 7,214,663, 7,223,837, 7,304,032, 7,329,507, 7,329,760,7,388,026, 7,655,660, 7,655,661, 7,906,545, and 8,012,978. Examples ofthe structures of the conjugate of the antibody-CC-1065 analogs via thebridge linker are as the following CC01, CC02, and CC03.

Wherein mAb is an antibody; n is 1˜20 Z₄ and Z′₄ are independently H,PO(OM₁)(OM₂), CH₂PO(OM₁)(OM₂), SO₃M₁, CH₃N(CH₂CH₂)₂NC(O)—,O(CH₂CH₂)₂NC(O)—, R₁, or glycoside; X₃ and X′₃ are independently O, NH,NHC(O), OC(O), —C(O)O, R₁, or absent; X₁, X₂, R₁, R₂, M₁, and M₂ are thesame defined in Formula (I).

Daunorubicin/Doxorubicin Analogues are also preferred for conjugationvia the bridge linkers of the present patent. The preferred structuresand their synthesis are exampled in: Hurwitz, E., et al., Cancer Res.35, 1175-1181 (1975). Yang, H. M., and Reisfeld, R. A., Proc. Natl.Acad. Sci. 85, 1189-1193 (1988); Pietersz, C. A., E., et al., E., etal.,” Cancer Res. 48, 926-9311 (1988); Trouet, et al., 79, 626-629(1982); Z. Brich et al., J. Controlled Release, 19, 245-258 (1992); Chenet al., Syn. Comm., 33, 2377-2390, 2003; King et al., Bioconj. Chem.,10, 279-288, 1999; King et al., J. Med. Chem., 45, 4336-4343, 2002;Kratz et al., J Med Chem. 45, 5523-33. 2002; Kratz et al., Biol PharmBull. January 21, 56-61, 1998; Lau et al., Bioorg. Med. Chem. 3,1305-1312, 1995; Scott et al., Bioorg. Med.l Chem. Lett. 6, 1491-1496;1996; Watanabe et al., Tokai J. Experimental Clin. Med. 15, 327-334,1990; Zhou et al., J. Am. Chem. Soc. 126, 15656-7, 2004; WO 01/38318;U.S. Pat. Nos. 5,106,951; 5,122,368; 5,146,064; 5,177,016; 5,208,323;5,824,805; 6,146,658; 6,214,345; 7569358; 7,803,903; 8,084,586;8,053,205. Examples of the structures of the conjugate of theantibody-CC-1065 analogs via the bridge linker are as the followingDa01, Da02, Da03 and Da04.

Wherein mAb is an antibody; n is 1˜20; X₃ and X′₃ are independently H,O, NH, NHC(O), NHC(O)NH, C(O), R₁, or OC(O); X₁, X₂, R₁, and R₂ are thesame defined in Formula (I).

Auristatins and dolastatins are preferred in conjugation via the bridgelinkers of this patent. The auristatins (e. g. auristain E (AE)auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E(MMAE), Monomethylauristatin (MMAF), Auristatin F phenylene diamine(AFP) and a phenylalanine variant of MMAE) which are synthetic analogsof dolastatins, are described in Int. J. Oncol. 15:367-72 (1999);Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 921-932 (2004); U.S.application Ser. Nos. 11/134,826, 20060074008, 2006022925. U.S. Pat.Nos. 4,414,205, 4,753,894, 4,764,368, 4,816,444, 4,879,278, 4,943,628,4,978,744, 5,122,368, 5,165,923, 5,169,774, 5,286,637, 5,410,024,5,521,284, 5,530,097, 5,554,725, 5,585,089, 5,599,902, 5,629,197,5,635,483, 5,654,399, 5,663,149, 5,665,860, 5,708,146, 5,714,586,5,741,892, 5,767,236, 5,767,237, 5,780,588, 5,821,337, 5,840,699,5,965,537, 6,004,934, 6,033,876, 6,034,065, 6,048,720, 6,054,297,6,054,561, 6,124,431, 6,143,721, 6,162,930, 6,214,345, 6,239,104,6,323,315, 6,342,219, 6,342,221, 6,407,213, 6,569,834, 6,620,911,6,639,055, 6,884,869, 6,913,748, 7,090,843, 7,091,186, 7,097,840,7,098,305, 7,098,308, 7,498,298, 7,375,078, 7,462,352, 7,553,816,7,659,241, 7,662,387, 7,745,394, 7,754,681, 7,829,531, 7,837,980,7,837,995, 7,902,338, 7,964,566, 7,964,567, 7,851,437, 7,994,135.Examples of the structures of the conjugate of the antibody-auristatinsvia the bridge linker are as the following Au01, Au02, Au03, Au04, andAu05.

Wherein mAb is an antibody; n is 1˜20; X₃ and X′₃ are independently CH₂,O, NH, NHC(O), NHC(O)NH, C(O), OC(O) R₁, or absent; X₄ and X′₄ areindependently CH₂, C(O), C(O)NH, C(O)N(R₁), R₁, NHR₁, NR₁, C(O)R₁ orC(O)O; Z₃ and Z′₃ are independently H, R₁, OP(O)(OM₁)(OM₂), NHR₁,OCH₂OP(O)(OM₁)(OM₂), OSO₃M1, or O-glycoside (glucoside, galactoside,mannoside, glucuronoside, alloside, fructoside), NH-glycoside,S-glycoside, or CH₂-glycoside; X₁, X₂, R₁, R₂ and R₃ are the samedefined in Formula (I).

The benzodiazepine dimers (e. g. dimmers of pyrrolobenzodiazepine (PBD)or (tomaymycin), indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidinobenzodiazepines) which are preferred cytotoxic agentsaccording to the present invention are exampled in the art: U.S. Pat.Nos. 8,163,736; 8,153,627; 8,034,808; 7,834,005; 7,741,319; 7,704,924;7,691,848; 7,678,787; 7,612,062; 7,608,615; 7,557,099; 7,528,128;7,528,126; 7,511,032; 7,429,658; 7,407,951; 7,326,700; 7,312,210;7,265,105; 7,202,239; 7,189,710; 7,173,026; 7,109,193; 7,067,511;7,064,120; 7,056,913; 7,049,311; 7,022,699; 7,015,215; 6,979,684;6,951,853; 6,884,799; 6,800,622; 6,747,144; 6,660,856; 6,608,192;6,562,806; 6,977,254; 6,951,853; 6,909,006; 6,344,451; 5,880,122;4,935,362; 4,764,616; 4,761,412; 4,723,007; 4,723,003; 4,683,230;4,663,453; 4,508,647; 4,464,467; 4,427,587; 4,000,304; US patent appl.20100203007, 20100316656, 20030195196. Examples of the structures of theconjugate of the antibody-benzodiazepine dimers via the bridge linkerare as the following PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB08,PB09, PB10 and PB11.

Wherein mAb is an antibody; n is 1-20; X₃ and X′₃ are independently CH₂,O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR₃), R₁, NHR₁, NR₁, C(O)R₁or absent; X₄ and X′₄ are independently CH₂, C(O), C(O)NH, C(O)N(R₁),R₁, NHR₁, NR₁, C(O)R₁ or C(O)O; X₁, X₂, R₁, R₂ and R₃ are the samedefined in Formula (I). In addition, R₁ and/or R₂ can be absent.

The drugs/cytotoxic agents used for conjugation via a bridge linker ofthe present patent can be any analogues and/or derivatives ofdrugs/molecules described in the present patent. One skilled in the artof drugs/cytotoxic agents will readily understand that each of thedrugs/cytotoxic agents described herein can be modified in such a mannerthat the resulting compound still retains the specificity and/oractivity of the starting compound. The skilled artisan will alsounderstand that many of these compounds can be used in place of thedrugs/cytotoxic agents described herein. Thus, the drugs/cytotoxicagents of the present invention include analogues and derivatives of thecompounds described herein.

All references cited herein and in the examples that follow areexpressly incorporated by reference in their entireties.

EXAMPLES

The invention is further described in the following examples, which arenot intended to limit the scope of the invention. Cell lines describedin the following examples were maintained in culture according to theconditions specified by the American Type Culture Collection (ATCC) orDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Braunschweig, Germany (DMSZ), or The Shanghai Cell Culture Institute ofChinese Acadmy of Science, unless otherwise specified. Cell culturereagents were obtained from Invitrogen Corp., unless otherwisespecified. All anhydrous solvents were commercially obtained and storedin Sure-seal bottles under nitrogen. All other reagents and solventswere purchased as the highest grade available and used without furtherpurification. The preparative HPLC separations were performed withVarain PreStar HPLC. NMR spectra were recorded on Varian Mercury 400 MHzInstrument. Chemical shifts (.delta.) are reported in parts per million(ppm) referenced to tetramethylsilane at 0.00 and coupling constants (J)are reported in Hz. The mass spectral data were acquired on a WatersXevo QTOF mass spect equipped with Waters Acquity UPLC separationsmodule and Acquity TUV detector.

Example 1: tert-Butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate(34)

To 350 mL of anhydrous THF was added 80 mg (0.0025 mol) of sodium metaland triethylene glycol 2 (150.1 g, 1.00 mol) with stirring. After thesodium had completely dissolved, tert-butyl acrylate (24 mL, 0.33 mol)was added. The solution was stirred for 20 h at room temperature andneutralized with 8 mL of 1.0 M HCl. The solvent was removed in vacuo andthe residue was suspended in brine (250 mL) and extracted with ethylacetate (3×125 mL). The combined organic layers were washed with brine(100 mL) then water (100 mL), dried over sodium sulfate, and the solventwas removed. The resulting colorless oil was dried under vacuum to give69.78 g (76% yields) of product 34. ¹H NMR: 1.41 (s, 9H), 2.49 (t, 2H,J=6.4 Hz), 3.59-3.72 (m, 14H). ESI MS m/z− C₁₃H₂₅O₆(M−H), cacld. 277.17.found 277.20.

Example 2. tert-Butyl3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (35)

A solution of 34 (10.0 g, 35.95 mmol) in acetonitrile (50.0 mL) wastreated with pyridine (20.0 mL). A solution of tosyl chloride (7.12 g,37.3 mmol) in 50 mL acetonitrile was added dropwise via an additionfunnel over 30 minutes. After 5 h TLC analysis revealed that thereaction was complete. The pyridine hydrochloride that had formed wasfiltered off and the solvent was removed. The residue was purified onsilica gel by eluting from with 20% ethyl acetate in hexane to with neatethyl acetate to give 11.2 g (76% yield) of compound 35. ¹H NMR: 1.40(s, 9H), 2.40 (s, 3H), 2.45 (t, 2H, J=6.4 Hz), 3.52-3.68 (m, 14H), 4.11(t, 2H, J=4.8 Hz), 7.30 (d, 2H, J=8.0 Hz), 7.75 (d, 2H, J=8.0 Hz); ESIMS m/z+C₂₀H₃₃O₈S (M+H), cacld. 433.18. found 433.30.

Example 3. tert-Butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate(36)

To 50 mL of DMF was added tert-butyl3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate 35 (4.0 g, 9.25mmol) and sodium azide (0.737 g, 11.3 mmol) with stirring. The reactionwas heated to 80° C. After 4 h TLC analysis revealed that the reactionwas complete. The reaction was cooled to room temperature and quenchedwith water (25 mL). The aqueous layer was separated and extracted intoethyl acetate (3×35 mL). The combined organic layers were dried overanhydrous magnesium sulfate, filtered, and the solvent removed in vacuo.The crude azide (about 90% pure by TLC) was used without furtherpurification. ¹H NMR (CDCl₃): 1.40 (s, 9H), 2.45 (t, 2H, J=6.4 Hz), 3.33(t, 2H, J=5.2 Hz), 3.53-3.66 (m, 12H). ESI MS m/z+ C₁₃H₂₆N₃O₈ (M+H),cacld. 304.18. found 304.20.

Example 4. 13-Amino-4,7,10-trioxadodecanoic acid tert-butyl ester, 37;13-Amino-bis(4,7,10-trioxadodecanoic acid tert-Butyl Ester), 38

The crude azide material 36 (5.0 g, ˜14.84 mmol) was dissolved inethanol (80 mL) and 300 mg of 10% Pd/C was added. The system wasevacuated under vacuum and placed under 2 atm of hydrogen gas viahydrogenation reactor with vigorous stirring. The reaction was thenstirred overnight at room temperature and TLC showed that the startingmaterials disappeared. The crude reaction was passed through a short padof celite rinsing with ethanol. The solvent was removed and the aminepurified on silica gel using a mixture of methanol (from 5% to 15%) and1% triethylamine in methylene chloride as the eluant to give13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester 37 (1.83 g, 44%yield, ESI MS m/z+ C₁₃H₂₇NO₅ (M+H), cacld. 278.19. found 278.30) and13-amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester), 38 (2.58 g,32% yield, ESI MS m/z+ C₂₆H₅₂NO₁₀ (M+H), cacld. 538.35. found 538.40).

Example 5. 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid, HClsalt, 39

To 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester, 37 (0.80 g,2.89 mmol) in 30 mL of dioxane was 10 ml of HCl (36%) with stirring.After 0.5 h TLC analysis revealed that the reaction was complete, thereaction mixture was evaporated, and co-evaporated with EtOH andEtOH/Toluene to form the title product in HCl salt (>90% pure, 0.640 g,86% yield) without further purification. ESI MS m/z+ C₉H₂₀NO₅ (M+H),cacld. 222.12. found 222.20.

Example 6. 13-Amino-bis(4,7,10-trioxadodecanoic acid, HCl salt, 40

To 13-amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester), 38 (1.00g, 1.85 mmol) in 30 mL of dioxane was 10 ml of HCl (36%) with stirring.After 0.5 h TLC analysis revealed that the reaction was complete, thereaction mixture was evaporated, and co-evaporated with EtOH andEtOH/Toluene to form the title product in HCl salt (>90% pure, 0.71 g,91% yield) without further purification. ESI MS m/z+ C₁₈H₃₆NO₁₀ (M+H),cacld. 426.22. found 426.20.

Example 7. bis(2,5-dioxopyrrolidin-1-yl) but-2-ynedioate, 9

To but-2-ynedioic acid 8 (2.0 g, 17.54 mmol) in DMA (100 ml) was addedNHS (5.0 g, 43.4 mmol) and EDC (12.0 g, 62.5 mmol). The mixture wasstirred under dark overnight, evaporated and purified on SiO₂ columneluted with EtOAc/DCM (1:10) to afford the title compound 9 (4.10 g, 76%yield). ESI MS m/z+ C12H₉N₂O₈ (M+H), cacld. 309.03. found 309.20.

Example 8. 4,7-dioxodec-5-ynedioic acid, 15

To a stirred solution of bis(trimethylsilyl)acetylene (5.0 g, 29.34mmol) and iodine (0.37 g, 1.45 mmol) in dichloromethane (100 mL), wasadded succinyl chloride (18.11 g, 116.83 mmol) slowly in a dropwisemanner at 0° C. After addition, the mixture was allowed to stir at roomtemperature until complete conversion as indicated by TLC (˜2 h). Thereaction mixture was quenched with water (15 mL) and extracted withdichloromethane (3×70 mL). The combined extracts were washed with 15%solution of sodium thiosulphate, dried over anhydrous Na₂SO₄, andconcentrated in vacuo. The resulting product was purified by columnchromatography on silica gel (100-200 mesh, aqueous form, from 5% to 10%of H₂O in acetonitrile) to afford the pure title product (5.50 g, yield85%). ESI MS m/z− C₁₀H₉O₆ (M−H), cacld. 226.05. found 226.10.

Example 9.(R,R,S,S,R,4R,4′R)-5,5′-(((4,7-dioxodec-5-ynedioyl)bis(azanediyl))bis(4-hydroxy-3,1-phenylene))bis(4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)2-methylpentanoicacid), 79

The compound 9 (25 mg, 0.081 mmol) in THF (3.0 ml) was added(4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-amino-4-hydroxyphenyl)-2-methylpentanoicacid, 51 (Huang Y. et al, Med Chem. #44, 249^(th) ACS National Meeting,Denver, Colo., March 22-26, 2015; WO2014009774) (151 mg, 0.199 mmol) inTHF (4.0 ml) and buffer (4 ml, 100 mM Na₂HPO₄, pH 7.0). After stirred atRT for 4 h, the mixture was concentrated and purified with C-18preparative HPLC (250 mm×ID 20 mm), eluted with water/ethanol (90% waterto 50% water in 55 min, v=15 ml/min). The fractions containing theproduct were pooled, concentrated and crystallized with EtOH/Hexane toafford the title compound (73 mg, 53% yield). ESI MSm/z+C₈₆H₁₂₂N₁₂N_(a)O₂₀S₂ (M+Na), cacld. 1729.83. found 1730.10.

Example 10.14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioicacid, 86

3-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)propanoic acid, HCl salt, 39 (601mg, 2.33 mmol) in the mixture of THF (6 ml) and a buffer (150 mMNaH₂PO₄, pH 7.2, 4 ml) was addedbis(2,5-dioxopyrrolidin-1-yl)but-2-ynedioate, 9 (350 mg, 1.13 mmol).After stirred at RT under dark for 4 h, the mixture was concentrated andpurified with SiO₂ column, eluted with water/acetonitrile (1:9). Thefractions containing the product were pooled and concentrated to affordthe title compound (345 mg, 590 yield). ESI MS m/z− C₂₂H₃₆N₂O₁₂ (M−H),cacld. 519.22. found 519.30.

Example 11.13,18-bis(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)ethyl)-14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioicacid, 87

13-Amino-bis(4,7,10-trioxadodecanoic acid, HCl salt, 40 (650 mg, 1.40mmol) in the mixture of THF (6 ml) and a buffer (150 mM NaH₂PO₄, pH 7.2,4 ml) was added bis(2,5-dioxopyrrolidin-1-yl)but-2-ynedioate, 9 (190 mg,0.61 mmol). After stirred at RT under dark for 4 h, the mixture wasconcentrated and purified with C-18 preparative HPLC (250 mm×ID 30 mm),eluted with water/ethanol (90% water to 50% water in 55 min, v=35ml/min). The fractions containing the product were pooled andconcentrated to afford the title compound (287 mg, 51% yield). ESI MSm/z− C₄₀H₆₇N₂O₂₂ (M−H), cacld. 927.42. found 928.30.

Example 12. Bis(2,5-dioxopyrrolidin-1-yl)14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioate,88

To14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioicacid, 86 (340 mg, 0.653 mmol) in DMA (6 ml) was added NHS (225 mg, 1.96mmol) and EDC (401 mg, 2.08 mmol). The mixture was stirred under darkovernight, evaporated and purified on SiO₂ column eluted with EtOAc/DCM(5:1) to afford the title compound 88 (330 mg, 71% yield). ESI MS m/z+C₃₀H₄₃N₄O₁₆ (M+H), cacld. 715.26. found 715.20.

Example 13. Bis(2,5-dioxopyrrolidin-1-yl)13,18-bis(2-(2-(2-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropoxy)ethoxy)ethoxy)ethyl)-14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioate,89

To13,18-bis(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)ethyl)-14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioicacid, 87 (280 mg, 0.301 mmol) in DMA (6 ml) was added NHS (105.0 mg,0.913 mmol) and EDC (200 mg, 1.04 mmol). The mixture was stirred underdark overnight, evaporated and purified on SiO₂ column eluted withEtOH/DCM (1:10-1:5) to afford the title compound 89 (249 mg, 63% yield).ESI MS m/z+ C₅₆H₈₁N₆O₃₀ (M+H), cacld. 1317.49. found 1317.80.

Example 14.(R,R,S,S,R,4R,4′R)-5,5′-(((14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioyl)bis(azanediyl))bis(4-hydroxy-3,1-phenylene))bis(4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methylpentanoicacid), 90

The compound 88 (30 mg, 0.042 mmol) in THF (3.0 ml) was added(4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-amino-4-hydroxyphenyl)-2-methylpentanoicacid, 51 (Huang Y. et al, Med Chem. #44, 249^(th) ACS National Meeting,Denver, Colo., Mar. 22˜26, 2015; WO2014009774) (80 mg, 0.107 mmol) inTHF (4.0 ml) and buffer (4 ml, 100 mM Na₂HPO₄, pH 7.0). After stirred atRT for 4 h, the mixture was concentrated and purified with C-18preparative HPLC (250 mm×ID 20 mm), eluted with water/ethanol (95% waterto 50% water in 55 min, v=15 ml/min). The fractions containing theproduct were pooled, concentrated and crystallized with EtOH/Hexane toafford the title compound (48 mg, 56% yield). ESI MS m/z−C₉₈H₁₄₇N₁₄O₂₆S₂(M−H), cacld. 2000.01. found 2000.40.

Example 15. Conjugated Compound 90 to an Antibody for 91

To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0˜8.0, were addedof 0.70˜2.0 mL PBS buffer of 100 mM NaH₂PO₄, pH 6.5˜7.5 buffers, TCEP(28 μL, 20 mM in water) and the compound 90 (14 μL, 20 mM in DMA). Themixture was incubated at RT for 2˜16 h, then DHAA (135 μL, 50 mM) wasadded in. After continuous incubation at RT overnight, the mixture waspurified on G-25 column eluted with 100 mM NaH₂PO₄, 50 mM NaCl pH6.0˜7.5 buffer to afford 16.8˜17.9 mg of the conjugate compound 91 (˜87%yield) in 13.1-14.9 ml buffer. The drug/antibody ratio (DAR) was 4.0(4.04), which was determined via UPLC-Qtof mass spectrum. It was 96˜99%monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mmID×30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGEgel.

Example 16. Compound 92 (Containing 4 Tubulysin Analogs Per BridgeLinker)

The compound 89 (35 mg, 0.026 mmol) in THF (3.0 ml) was added(4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-amino-4-hydroxyphenyl)-2-methylpentanoicacid, 51 (Huang Y. et al, Med Chem. #44, 249^(th) ACS National Meeting,Denver, Colo., Mar. 22˜26, 2015; WO2014009774) (100.6 mg, 0.132 mmol) inTHF (4.0 ml) and buffer (4 ml, 100 mM Na₂HPO₄, pH 7.0). After stirred atRT for 4 h, the mixture was concentrated and purified with C-18preparative HPLC (250 mm×ID 20 mm), eluted with water/ethanol (95% waterto 50% water in 50 min, v=15 ml/min). The fractions containing theproduct were pooled, concentrated and crystallized with EtOH/Hexane toafford the title compound 92 (47.6 mg, 47% yield). ESI MS m/z−C₁₉₂H₂₉₁N₂₆O₅₀S₄(M−H), cacld. 3890.00. found 3890.30.

Example 17. Conjugated Compound 92 to an Antibody for 93

To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0˜8.0, were addedof 0.70˜2.0 mL PBS buffer of 100 mM NaH₂PO₄, pH 6.5˜7.5 buffers, TCEP(28 μL, 20 mM in water) and the compound 92 (14 μL, 20 mM in DMA). Themixture was incubated at RT for 2˜16 h, then DHAA (135 μL, 50 mM) wasadded in. After continuous incubation at RT overnight, the mixture waspurified on G-25 column eluted with 100 mM NaH₂PO₄, 50 mM NaCl pH6.0˜7.5 buffer to afford 16.9˜17.5 mg of the conjugate compound 92 (˜85%yield) in 13.1˜14.9 ml buffer. The drug/antibody ratio (DAR) was 8.0(7.95), which was determined via UPLC-Qtof mass spectrum. It was 96˜99%monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mmID×30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGEgel.

Example 18. In Vitro Cytotoxicity Evaluation of Conjugates 91 and 93 inComparison with T-DM1

The cell lines used in the cytotoxicity assays were HL-60, a humanpromyelocytic leukemia cell line; NCI-N87, a human gastric carcinomacell line; BT-474, a human invasive ductal carcinoma cell line; andSKOV3, a human ovarian carcinoma cell line. For HL-60, NCI-N87, andBT-474 cells, the cells were grown in RPMI-1640 with 10% FBS. For SKOV3cells, the cells were grown in McCoy's 5A Medium with 10% FBS. To runthe assay, the cells (180 μl, 6000 cells) were added to each well in a96-well plate and incubated for 24 hours at 37° C. with 5% CO₂. Next,the cells were treated with test compounds (20 μl) at variousconcentrations in appropriate cell culture medium (total volume, 0.2mL). The control wells contain cells and the medium but lack the testcompounds. The plates were incubated for 120 hours at 37° C. with 5%CO₂. MTT (5 mg/ml) was then added to the wells (20 μl) and the plateswere incubated for 1.5 hr at 37° C. The medium was carefully removed andDMSO (180 μl) was added afterward. After it was shaken for 15 min, theabsorbance was measured at 490 nm and 570 nm with a reference filter of620 nm. The inhibition % was calculated according to the followingequation: inhibition %=[1−(assay−blank)/(control−blank)]×100.

The cytotoxicity results:

IC₅₀ ( nM) N87 cell (Ag+) SK-OV-3 cell (Ag+

HL60 cell (Ag

Conjugate 91 0.108 nM 0.089 nM >20 nM Conjugate 93 0.037 nM 0.029 nM >10nM T-DM1 0.270 nM 0.191 nM >15 nM

indicates data missing or illegible when filed

Specificity of conjugate 91 for N87 cell was over 185(IC₅₀>20/IC₅₀=0.108), and for SK-OV-3 cell was over 225; Specificity ofconjugate 93 for N87 cell was over 270 (IC₅₀>10/IC₅₀=0.037), and forSK-OV-3 cell was over 344; Specificity of conjugate T-DM1 for N87 cellwas over 55 (IC₅₀>15/IC₅₀=0.27), and for SK-OV-3 cell was over 78.

Both conjugate 91 and conjugate 93 were extremely more potent than thecommercial conjugate T-DM1. The conjugate 93 having DAR=8 was three-foldmore potent than conjugate 91 having DAR=4.

Example 19. Antitumor Activity In Vivo

The in vivo efficacy of conjugates 91 and 93 along with T-DM1 wereevaluated in a human gastric carcinoma N-87 cell line tumor xenograftmodels. Five-week-old female BALB/c Nude mice (24 animals) wereinoculated subcutaneously in the area under the right shoulder with N-87carcinoma cells (5×10⁶ cells/mouse) in 0.1 mL of serum-free medium. Thetumors were grown for 8 days to an average size of 130 mm³. The animalswere then randomly divided into 4 groups (6 animals per group). Thefirst group of mice served as the control group and was treated with thephosphate-buffered saline vehicle. The remaining three groups weretreated with conjugates 91, 93 and T-DM1 respectively at dose of 5 mg/Kgadministered intravenously. Three dimensions of the tumor were measuredevery 4 days and the tumor volumes were calculated using the formulatumor volume=1/2 (length x width x height). The weight of the animalswas also measured at the same time. A mouse was sacrificed when any oneof the following criteria was met: (1) loss of body weight of more than20% from pretreatment weight, (2) tumor volume larger than 1500 mm³, (3)too sick to reach food and water, or (4) skin necrosis. A mouse wasconsidered to be tumor-free if no tumor was palpable.

The results were plotted in FIG. 11. All the three conjugates compoundsdid not cause the animal body weight loss. And the animals at controlgroup were sacrificed at day 38 due to the tumor volume larger than 1500mm³ and all control animals were too sick. All 6/6 animals at the groupof compound 93 had completely no tumor measurable at day 14˜18 till day60 (the end of experiment). All 6/6 animals at the group of Compound 91group had no tumor measurable at day 14˜22 and 2/6 animals had tumorgrowth (measurable) back at days 42 and 50. In contrast only 2/6 animalsat the group of T-DM1 had no tumor measurable at days 14 and 22 untilday 38 and 50.

1. A compound of Formula (IV):

wherein: the acetylenedicarboxyl group in the bridge linker compound iscapable of reacting with a pair of sulfur atoms of a cell-binding agent;Drug₁ and Drug₂ are the same or different and represent a cytotoxicagent; R₁ and R₂ are the same or different, and are absent, linear alkylhaving from 1-6 carbon atoms; branched or cyclic alkyl having from 3 to6 carbon atoms; linear, branched or cyclic alkenyl or alkynyl havingfrom 3 to 6 carbon atoms; ester, ether, or amide having from 2 to 6carbon atoms; or polyethyleneoxy unit of formula (OCH₂CH₂)_(p), whereinp is an integer from 1 to about 1000, or combination thereof; or R₁ andR₂ are respectively a chain of atoms selected from C, N, O, S, Si, andP, which covalently connects to X₁ or X₂ and Z₁ or Z₂; and X₁ and X₂ areindependently NH, N(R₃), O, S or CH₂; R₃ is H, linear alkyl having from1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbonatoms, linear, branched or cyclic alkenyl or alkynyl, ester, ether, oramide having from 2 to 6 carbon atoms; or polyethyleneoxy unit offormula (OCH₂CH₂)_(p), wherein p is an integer from 1 to about 1000, orcombination thereof.
 2. The compound according to claim 1, wherein Drug₁and Drug₂ are the same or different and are independently selected fromthe group consisting of: 1). chemotherapeutic agents comprising: a).Alkylating agents comprising: Nitrogen mustards comprising:chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, mannomustine, mitobronitol, melphalan, mitolactol,pipobroman, novembichin, phenesterine, prednimustine, thiotepa,trofosfamide, and uracil mustard; CC-1065 and adozelesin, carzelesin andbizelesin synthetic analogues thereof; duocarmycin and syntheticanalogues thereof, KW-2189 and CBI-TMI; benzodiazepine dimers comprisingdimmers of pyrrolobenzodiazepine (PBD) or tomaymycin,indolinobenzodiazepines, imidazobenzothiadiazepines, andoxazolidinobenzodiazepines; Nitrosoureas comprising: carmustine,lomustine, chlorozotocin, fotemustine, nimustine, and ranimustine;Alkylsulphonates comprising: busulfan, treosulfan, improsulfan andpiposulfan; Triazenes; Platinum containing compounds comprising:carboplatin, cisplatin, and oxaliplatin; aziridines comprisingbenzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphaoramide andtrimethylolomelamine; b). Plant Alkaloids comprising: Vinca alkaloidscomprising: vincristine, vinblastine, vindesine, vinorelbine, andnavelbin; Taxoids comprising: paclitaxel, docetaxol and analogs thereof,Maytansinoids comprising DM1, DM2, DM3, DM4, DM5, DM6, DM7, maytansineand ansamitocins and analogs thereof, cryptophycins comprisingcryptophycin 1 and cryptophycin 8; epothilones, eleutherobin,discodermolide, bryostatins, dolostatins, auristatins, tubulysins, andcephalostatins; pancratistatin; a sarcodictyin; and spongistatin; c).DNA Topoisomerase inhibitors comprising: Epipodophyllins comprising:9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide,etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoicacids, teniposide, topotecan, and 9-nitrocamptothecin (RFS 2000);mitomycins; d). Antimetabolites comprising: Anti-folate: DHFR inhibitorscomprising: methotrexate, trimetrexate, denopterin, pteropterin, andaminopterin (4-aminopteroic acid); IMP dehydrogenase inhibitorscomprising: mycophenolic acid, tiazofurin, ribavirin, and EICAR;Ribonucleotide reductase inhibitors comprising: hydroxyurea, anddeferoxamine; Pyrimidine analogs comprising: Uracil analogs comprising:ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil,floxuridine, and ratitrexed (Tomudex); Cytosine analogs comprising:cytarabine, cytosine arabinoside, and fludarabine; Purine analogscomprising: azathioprine, fludarabine, mercaptopurine, thiamiprine, andthioguanine; folic acid replenisher; e). Hormonal therapies comprising:Receptor antagonists comprising: Anti-estrogen comprising: megestrol,raloxifene, and tamoxifen; LHRH agonists comprising: goscrclin, andleuprolide acetate; Anti-androgens comprising: bicalutamide, flutamide,calusterone, dromostanolone propionate, epitiostanol, goserelin,leuprolide, mepitiostane, nilutamide, testolactone, and trilostane;Retinoids/Deltoids comprising: Vitamin D3 analogs comprising: CB 1093,EB 1089 KH 1060, cholecalciferol, and ergocalciferol; Photodynamictherapies comprising: verteporfin, phthalocyanine, photosensitizer Pc4,and demethoxy-hypocrellin A; Cytokines comprising: Interferon-alpha,Interferon-gamma, tumor necrosis factor (TNFs), and human proteinscontaining a TNF domain; f). Kinase inhibitors comprising BIBW 2992(anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib.vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534),bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib,iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib,bevacizumab, cetuximab, Trastuzumab, Ranibizumab, Panitumumab, andispinesib; g). antibiotics comprising enediyne antibiotics comprisingcalicheamicins comprising calicheamicin γ1, δ1, α1 and β1; dynemicincomprising dynemicin A and deoxydynemicin; esperamicin, kedarcidin,C-1027, maduropeptin, and neocarzinostatin chromophore, aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin; chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, nitomycins, mycophe-nolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, and zorubicin; f). Polyketides (acetogenins) comprisingbullatacin and bullatacinone; gemcitabine, epoxomicins, bortezomib,thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032,STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy,Isoprenylation inhibitors, Dopaminergic neurotoxins, Cell cycleinhibitors, Actinomycins comprising Actinomycin D, and dactinomycin,Bleomycins comprising bleomycin A2, bleomycin B2, and peplomycin,Anthracyclines comprising daunorubicin, doxorubicin (adriamycin),idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDRinhibitors, Ca²⁺ ATPase inhibitors, Histone deacetylase inhibitorscomprising Vorinostat, Romidepsin, Panobinostat, Valproic acid,Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939,Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, and TrichostatinA; Thapsigargin, Celecoxib, glitazones, epigallocatechin gallate,Disulfiram, Salinosporamide A.; Anti-adrenals comprisingaminoglutethimide, mitotane, and trilostane; aceglatone; aldophosphamideglycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;eflornithine (DFMO), elfomithine; elliptinium acetate, etoglucid;gallium nitrate; gacytosine, hydroxyurea; ibandronate, lentinan;lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes comprising T-2 toxin,verrucarin A, roridin A and anguidine; urethane, siRNA, and antisensedrugs; 2). anti-autoimmune disease agents comprising: cyclosporine,cyclosporine A, aminocaproic acid, azathioprine, bromocriptine,chlorambucil, chloroquine, cyclophosphamide, corticosteroids comprisingamcinonide, betamethasone, budesonide, hydrocortisone, flunisolide,fluticasone propionate, fluocortolone danazol, dexamethasone,Triamcinolone acetonide, and beclometasone dipropionate, DHEA,enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate,mofetil, mycophenylate, prednisone, sirolimus, and tacrolimus; 3).anti-infectious disease agents comprising: a). Aminoglycosidescomprising: amikacin, astromicin, gentamicin comprising netilmicin,sisomicin, and isepamicin, hygromycin B, kanamycin comprising amikacin,arbekacin, bekanamycin, dibekacin, and tobramycin, neomycin comprisingframycetin, paromomycin, and ribostamycin, netilmicin, spectinomycin,streptomycin, tobramycin, verdamicin; b). Amphenicols comprising:azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol; c).Ansamycins comprising: geldanamycin, and herbimycin; d). Carbapenemscomprising: biapenem, doripenem, ertapenem, imipenem/cilastatin,meropenem, and panipenem; e). Cephems comprising: carbacephem,cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine,cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole,cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin,cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin,cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir,cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin,cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome,cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime,ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin comprisingcefoxitin, cefotetan, and cefmetazole, oxacephem comprising flomoxef,and latamoxef; f). Glycopeptides comprising: bleomycin, vancomycincomprising oritavancin, and telavancin, teicoplanin, and ramoplanin; g).Glycylcyclines; h). β-Lactamase inhibitors comprising: penam comprisingsulbactam, and tazobactam, and clavam; i). Lincosamides comprising:clindamycin, and lincomycin; j). Lipopeptides comprising: daptomycin,A54145, and calcium-dependent antibiotics (CDA); k). Macrolidescomprising: azithromycin, cethromycin, clarithromycin, dirithromycin,erythromycin, flurithromycin, josamycin, ketolide comprisingtelithromycin, and cethromycin, midecamycin, miocamycin, oleandomycin,rifamycins comprising rifampicin, rifampin, rifabutin, and rifapentine,rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus(FK506), troleandomycin, and telithromycin; 1). Monobac-tams: comprisingaztreonam, and tigemonam; m). Oxazolidinones; n). Penicillinscomprising: amoxicillin, ampicillin comprising pivampicillin,hetacillin, bacampicillin, metampicillin, and talampicillin,azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin,benzathine phenoxymethylpenicillin, clometocillin, procainebenzylpenicillin, carbenicillin, cloxacillin, dicloxacillin, epicillin,flucloxacillin, mecillinam, mezlocillin, meticillin, nafcillin,oxacillin, penamecillin, penicillin, pheneticillin,phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin,temocillin, and ticarcillin; o). Polypeptides comprising: bacitracin,colistin, and polymyxin B; p). Quinolones comprising: alatrofloxacin,balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin,enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin,grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin,marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin,ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin,sparfloxacin, temafloxacin, tosufloxacin, and trovafloxacin; q).Streptogramins comprising: pristinamycin, quinupristin and dalfopristin;r). Sulfonamides comprising: mafenide, prontosil, sulfacetamide,sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole,trimethoprim, and trimethoprim-sulfamethoxazole (co-trimoxazole); s).Steroid antibacterials; t). Tetracyclines comprising: doxycycline,chlortetracycline, clomocycline, demeclocycline, lymecycline,meclocycline, metacycline, minocycline, oxytetracycline,penimepicycline, rolitetracycline, tetracycline, and glycylcyclines; u).antibiotics comprising: annonacin, arsphenamine, bactoprenol inhibitors,DADAL/AR inhibitors, dictyostatin, discodermolide, eleutherobin,epothilone, ethambutol, etoposide, faropenem, fusidic acid,furazolidone, isoniazid, laulimalide, metronidazole, mupirocin,mycolactone, NAM synthesis inhibitors, nitrofurantoin, paclitaxel,platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin(rifampin), tazobactam tinidazole, and uvaricin; 4). anti-viral drugscomprising: a). Entry/fusion inhibitors comprising: aplaviroc,maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, and CD4(ibalizumab); b). Integrase inhibitors comprising: raltegravir,elvitegravir, and globoidnan A; c). Maturation inhibitors comprising:bevirimat, and vivecon; d). Neuraminidase inhibitors comprising:oseltamivir, zanamivir, and peramivir; e). Nucleosides and nucleotidescomprising: abacavir, aciclovir, adefovir, amdoxovir, apricitabine,brivudine, cidofovir, clevudine, dexelvucitabine, didanosine (ddI),elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil(5-FU), 3′-fluoro-substituted 2′, 3′-dideoxynucleoside analogues,fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), 1-nucleosidescomprising β-1-thymidine and β-1-2′-deoxycytidine, penciclovir, racivir,ribavirin, stampidine, stavudine (d4T), taribavirin (viramidine),telbivudine, tenofovir, trifluridine valaciclovir, valganciclovir,zalcitabine (ddC), and zidovudine (AZT); f). Non-nucleosides comprising:amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine,rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet(phosphonoformic acid), imiquimod, interferon alfa, loviride,lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa,podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), andtromantadine; g). Protease inhibitors comprising: amprenavir,atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir,nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), andtipranavir; h). anti-virus drugs comprising: abzyme, arbidol, calanolidea, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate(EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea,KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, andseliciclib; 5). a radioisotope selected from the group consisting of(radionuclides) ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, and ²¹³Bi; 6). achromophore molecule, which is capable of absorbing a UV light,florescent light, IR light, near IR light, or visual light; a class orsubclass of xanthophores, erythrophores, iridophores, leucophores,melanophores, cyanophores, fluorophore molecules which are fluorescentchemical compounds re-emitting light upon light, visualphototransduction molecules, photophore molecules, luminescencemolecules, luciferin compounds; Non-protein organic fluorophorescomprising: Xanthene derivatives comprising fluorescein, rhodamine,Oregon green, eosin, and Texas red; Cyanine derivatives comprising:cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, andmerocyanine; Squaraine derivatives and ring-substituted squarainescomprising Seta, SeTau, and Square dyes; Naphthalene derivativescomprising dansyl and prodan derivatives; Coumarin derivatives;Oxadiazole derivatives comprising pyridyloxazole, nitrobenzoxadiazoleand benzoxadiazole; Anthracene derivatives comprising anthraquinonescomprising DRAQ5, DRAQ7 and CyTRAK Orange; Pyrene derivatives; Oxazinederivatives comprising Nile red, Nile blue, cresyl violet, oxazine 170;Acridine derivatives comprising proflavin, acridine orange, and acridineyellow; Arylmethine derivatives comprising auramine, crystal violet, andmalachite green; Tetrapyrrole derivatives comprising porphin,phthalocyanine, and bilirubin; analogs and derivatives of fluorophorecompounds comprising: CF dye (Biotium), DRAQ and CyTRAK probes(BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLightFluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich),FluoProbes (Interchim), Abberior Dyes (Abberior), DY and MegaStokes Dyes(Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTauand Square Dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (BiosearchTechnologies), SureLight Dyes (APC, RPEPerCP, Phycobilisomes)(ColumbiaBiosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), Allophycocyanin(APC), Aminocoumarin, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2,Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin,Lissamine Rhodamine B, Lucifer yellow, Methoxycoumarin, NBD, PacificBlue, Pacific Orange, PE-Cy5 conjugates, PE-Cy7 conjugates, PerCP,R-Phycoerythrin(PE), Red 613, Seta-555-Azide, Seta-555-DBCO,Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-780-NHS, Seta-APC-780,Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405-Maleimide,SeTau-405-NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed,X-Rhodamine, 7-AAD (7-aminoactinomycin D, CG-selective), AcridineOrange, Chromomycin A3, CyTRAK Orange (Biostatus, red excitation dark),DAPI, DRAQ5, DRAQ7, Ethidium Bromide, Hoechst33258, Hoechst33342, LDS751, Mithramycin, Propidiumlodide (PI), SYTOX Blue, SYTOX Green, SYTOXOrange, Thiazole Orange, TO-PRO: Cyanine Monomer, TOTO-1, TO-PRO-1,TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. The fluorophore compounds that canbe linked to the linkers of the invention for study cells are selectedfrom the following compounds or their derivatives: DCFH(2′7′Dichorodihydro-fluorescein, oxidized form), DHR (Dihydrorhodamine123, oxidized form, light catalyzes oxidation), Fluo-3 (AM ester.pH >6), Fluo-4 (AM ester. pH 7.2), Indo-1 (AM ester, low/high calcium(Ca2+)), SNARF(pH 6/9), Allophycocyanin(APC), AmCyan1 (tetramer), AsRed2(tetramer), Azami Green (monomer), Azurite, B-phycoerythrin(BPE),Cerulean, CyPet, DsRed monomer, DsRed2 (“RFP”), EBFP, EBFP2, ECFP, EGFP(weak dimer), Emerald (weak dimer), EYFP (weak dimer), GFP (S65Amutation), GFP (S65C mutation), GFP (S65L mutation), GFP (S65Tmutation), GFP (Y66F mutation), GFP (Y66H mutation), GFP (Y66Wmutation), GFPuv, HcRed1, J-Red, Katusha, Kusabira Orange (monomer),mCFP, mCherry, mCitrine, Midoriishi Cyan (dimer), mKate (TagFP635,monomer), mKeima-Red (monomer), mKO, mOrange, mPlum, mRaspberry, mRFP1(monomer), mStrawberry, mTFP1, mTurquoise2, P3 (phycobilisome complex),Peridinin Chlorophyll (PerCP), R-phycoerythrin(RPE), T-Sapphire, TagCFP(dimer), TagGFP (dimer), TagRFP (dimer), TagYFP (dimer), tdTomato(tandem dimer), Topaz, TurboFP602 (dimer), TurboFP635 (dimer), TurboGFP(dimer), TurboRFP (dimer), TurboYFP (dimer), Venus, Wild Type GFP, YPet,ZsGreen1 (tetramer), and ZsYellow1 (tetramer); and 7). apharmaceutically acceptable salt, acid or derivative of any of the abovedrugs.
 3. The compound according to claim 1, wherein Drug₁ and Drug₂ area chromophore molecule.
 4. The compound according to claim 1, whereinDrug₁ and Drug₂ are selected from the group consisting of tubulysins,calicheamicins, auristatins, maytansinoids, CC-1065 analogs,daunorubicin and doxorubicin compounds, taxanoids (taxanes),cryptophycins, epothilones, benzodiazepine dimers comprising dimmers ofpyrrolobenzodiazepine (PBD), tomaymycin, anthramycin,indolinobenzodiazepines, imidazobenzothiadiazepines, andoxazolidinobenzodiazepines, calicheamicins and the enediyne antibiotics,actinomycin, azaserines, bleomycins, epirubicin, tamoxifen, idarubicin,dolastatins/auristatins comprising monomethyl auristatin E, MMAE, MMAF,auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP(AEFP), duocarmycins, thiotepa, vincristine, hemiasterlins, nazumamides,microginins, radiosumins, alterobactins, microsclerodermins,theonellamides, esperamicins, siRNA, nucleolytic enzymes, andpharmaceutically acceptable salts, acids, and analogues derivatives ofany of the above molecules.
 5. The compound according to claim 1,wherein Drug₁ and Drug₂ are a Tubulysin analog.
 6. The compoundaccording to claim 1, wherein Drug₁ and Drug₂ are a Calicheamicinanalog.
 7. The compound according to claim 1, wherein Drug₁ and Drug₂are a Maytansinoid analog.
 8. The compound according to claim 1, whereinDrug₁ and Drug₂ are a Taxane analog.
 9. The compound according to claim1, wherein Drug₁ and Drug₂ are a CC-1065 analogue or doucarmycin analog.10. The compound according to claim 1, wherein Drug₁ and Drug₂ are aDaunorubicin or Doxorubicin analogue.
 11. The compound according toclaim 1, wherein Drug₁ and Drug₂ are an Auristatin or dolastatinanalogue.
 12. The compound according to claim 1, wherein Drug₁ and Drug₂are a benzodiazepine dimer analogue.
 13. The compound according to claim1, wherein R₁ or R₂ is selected from the group consisting of6-maleimidocaproyl (MC), maleimido propanoyl (MP), valine-citrulline(val-cit), alanine-phenylalanine (ala-phe), lysine-phenylalanine(lys-phe), p-aminobenzyloxycarbonyl (PAB), 4-thio-pentanoate (SPP),4-(N-maleimidomethyl)cyclo-hexane-1-carboxylate (MCC), 4-thio-butyrate(SPDB), maleimidoethyl (ME), 4-thio-2-hydroxysulfonyl-butyrate(2-Sulfo-SPDB), pyridinyl-dithiol (PySS), alkoxy amino (AOA),ethyleneoxy (EO), 4-methyl-4-dithio-pentanoic (MPDP), azido (N₃),alkynyl, dithio, peptides, and (4-acetyl)aminobenzoate (SIAB).
 14. Thecompound according to claim 1, wherein R₁ or R₂ comprises a peptide of 1to 20 units of natural or unnatural amino acids, a p-aminobenzyl unit, a6-maleimidocaproyl unit, a disulfide unit, a thioether unit, a hydrozoneunit, a triazole unit, or an alkoxime unit.
 15. The compound accordingto claim 1, wherein R₁ or R₂ is cleavable by a protease.
 16. A methodfor preparing the compound according to claim 1, comprising reacting acompound of Formula (I) with a cell-binding molecule having a drugmolecule:

wherein Z₁, Z₂, R₁, R₂, X₁, and X₂ are defined the same as in claim 1.17. A method for preparing a cell-binding agent-drug conjugate, themethod comprising reacting the compound according to claim 1 with acell-binding molecule having a pair of free thiols.